COLUMBIA  LIBRARIES  OFFSITE 

HEALTH  SCIENCI  s  SIANDARD 


HX641401^n 
QP535.P1  F742  eestim 


RECAP 


m 


^- 


-fcs 


QP£Jt£jL 


ZZfi^ 


intl)fCitpofilruigork 

College  of  ^tpsiiciansJ  anb  burgeons 

Hibrarp 


Digitized  by  tine  Internet  Arciiive 

in  2010  with  funding  from 

Open  Knowledge  Commons  (for  the  Medical  Heritage  Library  project) 


http://www.archive.org/details/studiesonestimatOOforb 


.    Beetle  , 
D.  Wassow  , 


•oufMBiA  uNivrpsmr 
0€fA«TMENT  OF  PHYSIOLOG 

WtMT  fFTV^iKTM 


^ 


OHIO  AGRICULTURAL  EXPERIMENT  STATION 


J^ 


TECHNICAL  SERIES,  BULLETIN  No.  8 


STUDIES  ON  THE  ESTIMATION  OF 

INORGANIC  PHOSPHORUS  IN 

PLANT  AND  ANIMAL 

SUBSTANCES 


By  E.  B.  FORBES,  F.  M.  BEEGLE  AND  A.  F.  D.  WUSSOW 


WOOSTER,  OHIO,  U.  S.  A.,  JUNE,  1915 


OHIO  AGRICULTURAL  EXPERIMENT  STATION 

GOVERNING  BOARD 

THE  AGRICULTURAL  COMMISSION  OF  OHIO 

Columbus 


A.  P.  Sandles,  President 
S.  E.  Strode 


C.  G.  Williams 
H.  C.  Price 


B.  P.  Gayman, 

Secretary 


STATION  STAFF 

Charles  E.  Thorne,   M.   S.  A.,   Director 
DEPARTMENTAL   ORGANIZATION 


ADMINISTRATION 

The  Director,  Chief 

William  H.  Kramer,  Bursar 

W.   K.   Greenbank,   Librarian 

Horatio   Markley,   In  charge  of  Exhibits 

W.  J.  Holmes,  Printer 

Dora  Ellis,  Mailing  Clerk 

Glenn  Hall,  Engineer 

AGRONOMY 

C.   G.   Williams,   Chiefs 

P.  A.  Welton;  B.  S.,  Associate 

William  Holmes,  Farm  Manager 

C  A.  Patton,  Assistant 

C.  A.  Gearhart,  B.  S.,  Assistant 

E.  C.  MORR,  Office  Assistant 

C.  H.  Lebold,  Asst.  Foreman 

ANIMAL  HUSBANDRY 

B.  E.  Carjiichael,  M.  S.,  Chief 
J.  W.  Hammond,  M.  S.,  Associate 
Geo.  R.   Eastwood,   B.   S.,  Assistant 
Don  C.  Mote,   M.   S.,  Assistant 
W.  J.  Buss,  Assistant 
Anthony  Russ,  Herdsman 
E.  C.  SCHWAN,  Shepherd   (Carpenter) 

BOTANY 

A.  D.  Selby,  B.  S.,  Chief 

True  Housek,  B.  S.,  Asst.   (Germantown) 

P.   K.   Mathis,   Office  Assistant 

D.  C  Babcock,  A.  B.,  Assistant 
Richard  Walton,  B.   S..  Assistant 
J.  G.  Humbert,  B.  S.,  Assistant 

CHEMISTRY 

J.  W.  Ames,  M.  S.,  Chief 
Geo.  E.  Boltz,  B.  S.,  Assista7it 
J.  A.   Stenius,  B.   S.,  Assistant 

C.  J.   Schollenberger,  Assistant 
Mabel  K.  Corbould,  Assistant 
T.  E.  Richmond,  M.  S.,  Assistant 

CLIMATOLOGY 

J.    Warren    Smith,   Chief    (Columbus)^ 
C.  A.  Patton,  Observer 

DAIRYING 

C.  C.  Kayden,  M.  S.,  Chief 
A.   E.  Perkins,  M.  S.,  Assistant 
T.  R.  Middaugh,  Office  Assistant 

ENTOMOLOGY 

H.  a.  Gossard,  M.  S.,  Chief 
J.  S.  HousER,  M.  S.  A.,  Associate 
W.  H.  Goodwin,  M.  S.,  Assistant 
R.  D.  Whitmarsh,  M.  S.,  Assistant 
J.  L.  King,  B.  S.,  Assistant 

FORESTRY 

Edmund  Secrest,  B.  S.,  Chief 
J.  J.  Crumley,  Ph.  D.,  Assistant 
A.  E.  Taylor,  B.   S.,  Assistant 
J.  W.  Calland,  B.  S.,  Assistant 

D.  E.  Snyder,  Office  Assistant 
*With  leave  of  absence.    ^In  cooperation  with 


HORTICULTURE 

W.  J.  Green,   Vice  Director,  Chief 
P.  H.   Ballou,  Assistant    (iJewark) 
Paul  Thayer,  M.  S.,  Assistant 
C.  W.  Ellenwood,  Office  Assistant 
Ora  Flack,  Foreman  of  Orchards 
W.   E.  Bontrager,  Foremast  of  Grounds 
C.  G.  Laper,  Foreman  of  Greenhouses 
J.   B.  Keil,  Orchard  Assistant 
S.  N.  Green,  Garden  Assistant 

NUTRITION 

E.  B.  Forbes,  Ph.  D.,  Chief 
P.  M.  Beegle,  B.  S.,  Assistant 
Charles  M.  Fritz,  M.  S.,  Assistant 
L.  E.  Morgan,  M.  S.,  Assistant 
S.  N.  Rhue,  B.  S.,  Assistant 

SOILS 

The  Director,  Chief 

C.  G.  Williams,  Associate  in  soil  fertility 

investigations 
J.  W.  Ames,  M.  S.,  Associate  in  chemistry 

E.  R.    Allen,    Ph.    D.,    Associate   in    soil 
biology 

H.  Foley  Tuttle,  M.  S.,  Assistant 
A.   Bonazzi,   B.   Agr.,  Assistant 
John  Woodard,  M.  S.,  Assistant 

FARM  MANAGEMENT 

C.  W.  Montgomery,  Chief 

F.  N.  Meeker,  Executive  Assistant 

District  Experiment  Farms 

Northeastern  Test-Farm,  Strongsville. 

J.  Paul  Markley,  Resident  Manager 

Southwestern  Test-Farm,  Germantown 

Henry  M.  Wachter,  Resident  Manager 

Southeastern  Test-Farm,  Carpenter. 
H.   D.  Lewis,  Resident  Manager 

Northwestern  Test-Farm,  Findlay. 

John  A.  Sutton,  Resident  Manager 

County  Experiment  Farms 

Miami  County  Experiment  Farm,  Troy 
M.  C.  Thomas,  Agent  in  Charge 

Paulding  County  Experiment  Farm,  Paulding 

C.  Ellis  Bundy,  Agent  in  Charge 

Clermont  Co.  Experiment  Farm.  Owensville 

Victor  Herron,  Agent  in  Charge 
Hamilton  Co.  Experiment  Farm,  Mt.  Healthy 

D.  R.  Van  Atta,  B.  S.,  Agent  in  Charge 
Washington  County  Experiment  Farms, 

Fleming  and  Marietta 

E.  J.  RiGGS,  B.  S.,  Agent  in  Charge 
Mahoning  Co.  Experiment  Farm 

D.  W.  Galehouse,  Agent  in  Charge 
Trumbull  Co.  Experiment  Farm,  Cortland 
j\r.  O.  BUGBY,  B.  S.,  Agent  in  Charge 
Weather  Service,  U.  S.  Department  of  Agriculture. 


s 
o 


BULLETIN 

OF  THE 

Ohio  Agricultural  Experiment  Station 

Number  8  Technical  Semes  June,  1915 


STUDIES  ON  THE  ESTIMATION  OF  INORGANIC  PHOS- 
PHORUS IN  PLANT  AND  ANIMAL  SUBSTANCES 

BY  E.  B.  FORBES,  F.  M.  BEEGLE  AND  A.  F.  D.  WUSSOW 

In  connection  with  studies  of  the  metabolism  of  plants  and 
animals  it  is  frequently  desired  to  distinguish  between  simple  inor- 
ganic phosphates  and  phosphorus  in  combination  with  organic 
groups.  For  the  purpose  of  making  such  separate  estimations  there 
were  published  from  this  laboratory,  by  Forbes,  Lehmann,  Collison 
and  Whittier,  as  Ohio  Agr.  Exp.  Station  Bui.  215,  two  different 
methods  of  inorganic  phosphorus  determination,  one  recommended 
for  use  with  plant  substances  and  the  other  for  use  with  products 
of  animal  origin.  Since  issuing  this  publication,  five  years  ago, 
we  have  come  to  realize  that  the  problem  was  more  difficult  than  we 
had  known  it  to  be,  and  that  further  evidence  was  required  for  judg- 
ment as  to  the  correctness  of  our  analytical  procedures.  In  this 
spirit  we  have  done  further  work  on  these  methods,  the  result  being 
the  improvement  and  establishment  of  our  method  for  animal  sub- 
stances, and  the  demonstration  of  the  imperfection  of  our  method 
for  vegetable  products.  In  spite  of  the  considerable  measure  of 
failure  attending  our  efforts  to  determine  inorganic  phosphorus  in 
vegetable  substances,  illumination  of  the  problem  was  accomplished, 
and  on  this  account  we  report  the  results  of  this  study.  That  por- 
tion of  this  paper  which  deals  with  plant  substances  will  be  found 
on  pages  3  to  23,  and  that  portion  having  to  do  with  animal  pro- 
ducts on  pages  23  to  40. 

(3) 


4  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

THE  ESTIMATION  OF  INORGANIC  PHOSPHORUS  IN 
VEGETABLE  SUBSTANCES 

The  essential  points  of  the  method  of  Forbes  and  associates, 
as  published,  were  (1)  extraction  of  a  10-gram  sample  in  300  c.  c. 
of  0.2  percent  hydrochloric  acid  for  three  hours,  (2)  filtration,  and 
precipitation  of  a  250  c.  c.  portion  of  the  extract,  with  magnesia 
mixture  and  ammonia,  (3)  washing  the  precipitate  with  2.5  per- 
cent ammonia  and  with  95  percent  alcohol,  the  precipitate  being 
then  allowed  to  dry,  (4)  the  separation  of  the  inorganic  phosphates 
from  the  remainder  of  the  precipitate  by  breaking  up  the  paper  and 
precipitate  in  100  c,  c.  of  0.2  percent  nitric  acid  in  95  percent  alco- 
hol, (5)  filtration  of  the  extract,  pipetting  out  75  c.  c.  of  the  filtrate, 
(6)  evaporation  of  the  alcohol,  taking  up  with  0.2  percent  aqueous 
nitric  acid,  and  estimation  of  phosphorus  in  this  solution  by  taking 
through  the  molybdate  and  magnesia  mixture  precipitates,  and  esti- 
mation as  the  pyrophosphate  in  the  usual  way.  The  result  repre- 
sents 6.25  grams  out  of  the  original  10  grams  of  sample. 

The  evidence  upon  which  this  method  was  based  was  submitted 
in  the  original  publication.  In  the  further  study  of  this  method 
obsei'vations  were  made  as  will  be  discussed.  Our  bases  for  judg- 
ment as  to  the  correctness  of  the  method  were  the  agreement  of 
triplicates,  the  completeness  of  recovery  of  added  phosphates,  and 
the  completeness  of  extraction  during  the  three-hour  period  speci- 
fied, as  determined  by  subjection  of  the  residue  to  further 
extraction. 

In  the  further  study  of  this  method  especial  attention  was  given 
to  (1)  the  use  of  phenol  in  the  extractive  reagent,  (2)  the  complete- 
ness of  the  3-hour  extraction,  (3)  improvement  of  the  method  of 
filtration,  and  other  details  of  technique. 

In  the  original  publication  of  the  method  the  use  of  phenol 
in  the  extractive  reagent  was  suggested  in  cases  where  the  activity 
of  enzymes  seemed  to  aff'ect  results.  This  was  considered  especially 
likely  to  be  the  case  in  estimations  involving  difficult  and  therefore 
protracted  filtration,  since  it  was  found  that  the  slower  the  filtra- 
tion the  greater  would  be  the  amount  of  inorganic  phosphorus 
found. 

In  the  working  out  of  the  details  of  the  method  as  published, 
the  duration  of  the  extraction  period  received  some  consideration, 
and  the  15-minute  extraction  of  Hart  and  Andrews,  from  whose 
method  the  extractive  reagent  was  adopted,  was  in  our  method 
lengthened  to  three  hours;  but  our  fixing  upon  this  duration  of 
extraction  was  largely  arbitrary,  and  further  evidence  was  deemed 
desirable  to  establish  this  point  in  a  consistent  manner. 


INORGANIC  PHOSPHORUS  ESTIMATION  5 

Aqueous  extracts  of  vegetable  substances  are  often  exceedingly 
difficult  to  filter.  The  method  as  published  involved  much  difficult 
filtration,  and  with  some  vegetable  substances  was,  on  this  account, 
practically  unworkable.  In  the  studies  here  reported  these  diffi- 
culties of  filtration  were  removed. 

These  points  and  others  were  considered  in  a  series  of  analyti- 
cal determinations,  as  reported  in  the  following  tables,  the  work 
usually  including,  as  a  check,  a  test  of  the  completeness  of  recovery 
of  added  inorganic  phosphorus.  The  method,  as  used  in  this  work, 
was  as  follows : 

ACID-ALCOHOL  METHOD  OF  FORBES  AND  ASSOCIATES  FOR  THE 

DETERMINATION  OF  INORGANIC  PHOSPHORUS  IN 

VEGETABLE  SUBSTANCES 

Pour  exactly  300  c.  c.  of  0.2  percent  hydrochloric  acid  (4.6  c.  c.  concen- 
trated hydrochloric  acid,  sp.  gr.  1.18-1.19,  per  liter)  onto  10  grams  of  sample 
in  a  dry  400  c.  c.  Florence  flask.  Close  with  rubber  stopper,  and  shake  at  inter- 
vals of  5  minutes  for  3  hours.  Filter  the  extract  by  suction  into  dry  flasks 
through  S.  &  S.  No.  589  "Blue  Ribbon"  papers,  in  a  Witt  filtering  appai-atus,  or 
a  Biichner  funnel. 

Measure  out  a  250  c.  c.  portion  of  this  filtered  extract,  and  precipitate  in 
a  400  c.  c.  beaker  with  10  c.  c.  magnesia  mixture  and  20  c.  c.  ammonia,  sp.  gr. 
0.9.  Allow  to  stand  over  night,  and  filter  through  double  S.  &  S.  No.  589  "White 
Ribbon"  papers,  taking  care  to  decant  as  long  as  possible  without  pouring  out 
the  precipitate.     Then  complete  the  transfer  of  the  precipitate  to  the  paper. 

Wash  three  times  with  2.5  percent  ammonia,  and  then  three  times  with 
95  percent  alcohol.  Allow  the  precipitate  to  drain,  and  then  spread  out  the 
inner  paper  on  the  top  of  the  funnel,  and  allow  the  alcohol  to  evaporate.  When 
practically  dry,  place  this  inner  paper  with  the  precipitate  into  an  Erlenmeyer 
flask.  Add  100  c.  c.  of  95  percent  alcohol  containing  0.2  percent  of  nitric  acid. 
Close  the  flask  with  a  rubber  stopper  and  shake  vigorously  until  the  paper  is 
thoroughly  broken  up.  If  the  precipitate  is  flaky,  and  refuses  to  break  up  on 
shaking,  allow  to  stand  in  the  acid-alcohol  over  night. 

Now  filter  through  a  dry  filter  into  a  dry  flask.  Pipette  out  75  c.  c.  of  the 
filtrate  into  a  small  beaker,  and  evaporate  almost  but  not  quite  to  dryness! 
Dissolve  in  dilute  nitric  acid,  and  filter  if  necessary;  then  determine  phosphorus 
in  the  usual  gravimetric  way,  by  precipitation  first  with  acid  molybdate  solution, 
later  with  magnesia  mixture,  and  then  burning  to  the  pyrophosphate. 

The  result  obtained  as  above  represents  6.25  grams  out  of  .the  original 
10  grams  of  material,  and  so  to  reduce  to  a  1-gram  basis  multiply  by  0.16. 

In  the  analyses  reported  in  the  following  group  of  tables  (pages 
7  to  10)  the  above  standard  method  was  modified,  in  certain  parts 
of  the  work,  as  indicated  below ; 


6  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

AQUEOUS  HYDEOCHLORIC  ACID  EXTRACTION,  PLUS  PHOSPHATE 

Proceed  as  on  p.  5  except  that  in  place  of  300  c.  c.  of  0.2  percent  hydro- 
chloric acid  add  250  c.  c.  of  the  same  and  50  c.  c.  of  phosphate  solution  contain- 
ing disodium  phosphate  equivalent  to  approximately  25  mg.  magnesium  pyro- 
phosphate per  50  c.  c.  Make  up  this  phosphate  solution  with  0.2  percent 
hydrochloric  acid. 

AQUEOUS  HYDROCHLORIC  ACID-PHENOL  EXTRACTION 

Proceed  as  on  p.  5  except  that  in  place  of  300  c.  c.  of  0.2  percent  hydro- 
chloric acid  add  300  c.  c.  of  0.2  percent  hydrochloric  acid  solution  containing 
50  gm.  phenol  per  liter, 

AQUEOUS  HYDROCHLORIC  ACID-PHENOL  EXTRACTION,  PLUS  PHOSPHATE 

■  Proceed  as  in  the  paragraph  above  except  that  in  place  6f  300  <;.  c.  add 
250  c.  c.  of  0.2  percent  hydrochloric  acid  containing  50  gm.  phenol  per  liter, 
and  50  c.  c.  of  phosphate  solution  containing  disodium  phosphate  equivalent  to 
approximately  25  mg.  magnesium  pjTophosphate  per  50  c.  c.  Make  up  this 
phosphate  solution  with  0.2  percent  hydrochloric  acid  containing  50  gm.  phenol 
per  liter. 


PHOSPHORUS  ESTIMATIONS  ON  REAGENTS  AND  PHOSPHATE  SOLU- 
TIONS USED  IN  WORK  REPORTED  IN  TABLE  I,  PAGE  7 

Magnesium 
pj^rophosphate 


Blank  1  Aq.-HCl  solutions 

2  Aq.-HCl  solutions 

3  Aq.-HCl  solutions 

Average 

Blank  1  Aq.-HCl-phenol  solutions 

2  Aq.-HCl-phenol  solutions 

3  Aq.-HCl-phenol  solutions 

Average 

Phosphate  solution  (Aq.-HCl)  50  cc.  1 

Phosphate  solution  (Aq.-HCl)  50  cc.  2 

Phosphate  solution  (Aq.-ACl)  50  cc.  3 

Average 

Phosphate  solution  (Aq.-HCl-phenol)  50  cc.  1. 

Phosphate  solution  (Aq.-HCl-phenol)  50  cc-  2. 

Phosphate  solution  (Aq.-HCl-phenol)  50  cc.  3. 

Average 


0.0002 
0.0002 
0.0002 

0.0002 

0.0002 
0.0002 
0.0002 

0.0002 

0.0250 
0.0248 
0.0248 

0.0249 

0.0249 
0.0249 
0.0249 

0.0249 


INORGANIC  PHOSPHORUS  ESTIMATION  1 

TABLE  I:     TEST  OF  THE  ACID-ALCOHOL  ^lETHOD  OF  FORBES  AND 

ASSOCIATES  FOR  THE  DETERMINATION  OF  INORGANIC 

PHOSPHORUS  IN  VEGETABLE  SUBSTANCES 


Sample 


Number  ami  troatnient 


Alfalfa 


Ai  Aci.-HCl  extraction 

A2  Aq.-HCl  extraction 

Averajre 

Bi  Aq.-HCl  extr.  plus  phosphate 

B2  Aq.-HCl  e.xtr.  plus  phosphate 

Bs  Aq.-HCl  extr.  plus  phosphate.. 

Averaire 

Ci  Aq.-HCl-phenoIextraction 

C'j  Aq.-HCl-phenolextraction 

C3  Aq.-HCl-phenolextraction 

Average 

Di  Aq.-HCl-phenolextr.  plus  phosphate. 
D2  Aq.-HCl-phenol  extr.  plus  phosphate. 
Ds  Aq.-HCl-phenol  e.xtr.  plus  phosphate. 
Averag-e 


Magne- 
sium 
pyro- 
phos- 
phate 

Grams 


0.0189 
0.0170 
0.0180 
0.0188 
0.0222 
0.0225 
0.0212 
0.0182 
0.0168 
0.0175 
0.0175 
0.0210 
0.0203 
0.0229 
0.0213 


Inor- 
ganic 
phos- 
phorus 

Percent 


0.0803 


0.0780 


Phos- 
phorus 
added 
(magne- 
sium 
pyro- 
phos- 
phate) 

Grams 


0.0156 


0.0156 


Added 
phos- 
phorus 
recovered 
(magne 
slum 
pyro- 
phos- 
phate) 
Grams 


0.0032 


0.0038 


Added 
phos- 
phorus 
recovered 

Percent 


20.5 


24.3 


Blue 
Grass 


Al  Aq.-HCl  extraction 

A2  Aq.-HCl  extraction 

A3  Aq.-HCl  extraction 

Average 

Bi  Aq.-HCl  extr.  plus  phosphate. 

B2  Aq.-HCl  extr.  plus  phosphate 

B3  Aq.-HCl  extr.  plus  phosphate 

Average 

Ci  /r  q.-HCl-phenol  extraction 

C2  Aq.-HCl-phenol  extraction 

C3  Aq.-HCl-phenol  extraction. 

A  verage 

Di  Aq.-HCl-phenol  e.xtr.  plus  phosphate. 
D2  Aq.-HCl-phenol  extr.  plus  phosphate. 
D3  Aq.-HCl-phenol  extr.  plus  phosphate. 
Average 


0.0228 
0.0225 
0.0224 
0.0226 
0.0283 
0.0230 
0  0255 
0.0256 
0.0365 
0.0355 
0.0347 
0.0356 
0.0493 
0.0458 
0.0498 
0.0483 


0. 1008 


0. 1588 


0.0156 


0.0156 


Brewer's 
Grains 


Al  Aq.-HCl  extraction 

I A2  Aq.-HCl  extraction 

A3  A  q.-HCl  extraction 

Average 

Bi  Aq.-HCl  extr.  plus  phosphate : . 

B2  Aq.-HCl  extr.  plus  phosphate 

B3  Aq  -HClextr.  plus  phosphate 

Average 

Ci  Aq.-HCl-phenol  extraction 

C2  Aq.-HCl-phenol  extraction 

C3  Aq.-HCl-phenolextraction 

A  verage 

Di  Aq.-HCl-phenol  e.xtr.  plus  phosphate. 
D2  Aq.-HCl-phenol  e.xtr.  plus  phosphate. 
D3  Aq.-HCl-phenolextr.  plus  phosphate. 
Average 


0.0025 
0.0021 
0.0023 
0.0023 
0.0160 
0.0148 
0.0161 
0.0156 
0.0012 
0.0010 
0.0013 
0.0012 
0.0162 
0.0158 
0.0160 
0.0160 


Rice 
Polish 


Al  Aq.-HCl  extraction 

A2  Aq.-HCl  extraction 

A3  Aq.-HCl  extraction 

A  verage 

Bi  Aq.-HCl  extr.  plus  phosphate 

B2  Aq.-HCl  extr.  plus  phosphate 

Bs  Aq  -HCl  extr.  plus  phosphate 

Average 

Ci  Aq.-HCl-phenolextraction 

C2  Aq.-HCl-phenol  extraction 

C3  Aq.-HCl-phenol  extraction 

Average 

Di  Aq.-HCl-phenolextr.  plus  phosphate. 
U2  Aq.-HCl-phenolextr.  plus  phosphate. 
D3  Aq.-HCl-phenolextr.  plus  phosphate. 
A  verage 


0.0038 

0.0038  - 

0.0035 

0.0037 

0.0119 

0.0110 

0.0111 

0.0113 

0.0027 

0.0017 

0.0024 

0.0023 

0.0052 

0.0093 

0.0138 

0.0094 


0.0103 


0.0053 


0.0165 


0.0102 


0.0156 


0.0156 


0.0030 


0.0127 


19.2 


81.4 


0.0156 


0.0156 


0.0133 


0.0148 


0.0076 


0.0071 


85.2 


94.9 


48.7 


45.5 


8 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


TABLE  II:     TEST  OF  THE  ACID-ALCOHOL  METHOD  OF  FORBES  AND 

ASSOCIATES  FOR  THE  DETERMINATION  OF  INORGANIC 

PHOSPHORUS  IN  VEGETABLE  SUBSTANCES 


Sample 


Alfalfal 


Blue 

Grass^ 


Rice 
Polish  3 


Number  and  treatment 


Magne- 
sium 
pyro- 
phos- 
phate 

Grams 


Ai  Aq.-HCl  extraction 

A2  Aq.-HCl  extraction 

A3  Aq.-HCl  extraction 

Average 

Bi  Aq.-HCl  extr.  plus  phosphate 

B3  Aq.-HCl  extr.  plus  phosphate 

Average 

Cl  Aq.-HCl-phenol  extraction 

C2  Aq.-HCl-phenol  extraction 

Cs  Aq.-HCl-phenol  extraction 

Average 

Di  Aq.-HCl-phenol  extr.  plus  phosphate 
D2  Aq.-HCl-phenol  extr.  plus  phosphate 
Average 

Ai  Aq.-HCl  extraction 

A2  Aq.-HCl  extraction 

As  Aq.-HCl  extraction 

Average 

Bi  Aq.-HClextr.  plus  phosphate 

B2  Aq.-HCl  extr.  plus  phosphate 

Bs  Aq.-HCl  extr.  plus  phosphate 

Average 

Cl  Aq.-HCl-phenol  extraction 

C2  Aq.-HCl-phenol  extraction 

Ca  Aq.-HCl-phenol  e.xtraction 

Average 

Di  Aq.-HCl-phenol  extr.  plus  phosphate 
D2  Aq.-HCl-phenol  extr.  plus  phosphate 
Ds  Aq.-HCl-phenol  extr.  plus  phosphate 
Average 

Ai  Aq.-HCl  extraction 

A2  Aq.-HCl  extraction 

As  Aq.-HCl  extraction 

Average 

Bi  Aq.-HCl  extr.  plus  phosphate 

B2  Aq.-HCl  extr.  plus  phosphate 

Bs  Aq.-HCl  e.xtr.  plus  phosphate 

Average 

Cl  Aq.-HCl-phenol  extraction 

C2  Aq.-HCl-phenol  extraction 

Cs  Aq.-HCl-phenol  extraction 

Average 

Di  Aq.-HCl-phenol  extr.  plus  phosphate 
D2  Aq.-HCl-phenol  extr.  plus  phosphate 
Average 


0.0219 
0.0184 
0.0160 
0.0188 
0.0237 
0.02.35 
0.0236 
0.0140 
0.0189 
0.0149 
0.0159 
0.0232 
0.0241 
0.0236 


0.0380 
0.0379 
0.0335 
0.0365 
0.0321 
0.0298 
0.0320 
0.0313 
0.0397 
0.0409 
0.0408 
0.0404 
0.0554 
0.0548 
0.0545 
0.0549 


0.0036 
0.0026 
0.0027 
0.0030 
0.0076 
0.0075 
0.0079 
0.0077 
0.0020 
0.0020 
0.0020 
0.0020 
0.0064 
0.0082 
0.0073 


Inor- 
ganic 
phos- 
phorus 


0.0838 


0.0709 


0. 1628 


0. 1802 


0.0134 


0.0089 


Phos- 
phorus 
added 
(magne- 
sium 
pyro- 
phos- 
phate) 

Grams 


Added 
phos- 
phorus       .  J.    . 

recovered     Aaaea 

(magne 


Slum 
pyro- 
phos- 
phate) 

Grams 


phos- 
phorus 
recovered 

Percent 


0.0156 


0.0156 


0.0156 


0.0156 


0.0156 


0.0156 


0.0048 


0.0077 


30. 


49.3 


-0.0052 


0.0145 


-33.3 


92.9 


0.0047 


0.0053 


30.1 


34. 


(1)  Second  set  of  determinations;  first  magnesium  precipitates  allowed  to  stand  an  extra 
day  before  filtering,  and,  after  filtering,  an  extra  day  in  acid  alcohol.  With  samples  A-1  and 
A-3  only  200  c.  c.  of  aqueous-HCl  extract  was  used,  but  the  figures  given  represent  250  c.  c. 
as  usual. 

(2)  With  samples  A-1,  A-2  and  A-3  only  200  c.  c.  of  the  aqueous  HCl  extract  was  used, 
but  weights  given  for  magnesium  pyrophosphate  represent  250  c.   c. 

(3)  First  magnesium  precipitate  broken  up  in  acid  alcohol  with  stirring  rod  before 
filtering  oflf  75  c.  c.  aliquot. 


INORGANIC  PHOSPHORUS  ESTIMATION  9 

TABLE  III:     TEST  OF  COMPLETENESS  OF  EXTRACTION  AND  INFLU- 
ENCE OF  PHENOL  IN  THE  DETERMINATION  OF  INORGANIC 
PHOSPHORUS  IN  VEGETABLE  SUBSTANCES 


First  extraction 

Second  extraction 

Phos- 
phorus 

Added 

pho.s- 

Excess 

added 

phorus 

Mag'ne- 

phos- 
phorus 

Mag-ne- 

Inor- 

sium 

extracted 

Sample,  treatment 

sium 

g'anic 

slum 

pyro- 

(magrne- 

and  sample  number 

pyrophos- 

phos- 

phos- 
phate) 

pyro- 

phos- 

sium 

phate 

phorus 

phos- 

phate 

pyro- 
phos- 

phate) 

phate) 

Grams 

Percent 

Grams 

Grams 

Grams 

Grams 

Timothy:              Ai 

0.0064 

0.0012 

Aq.-HCl                 A2 

0.0069 

0.0010 

extraction            A3 

0.0053 

0.0012 

Av. 

0  0062 

0.0276 

0.0011 

-f  0.0001 

Timothy:              A  4 

0.0253 

0.0032 

Aq.-HClextr.          A;, 

0.0250 

0.0033 

plus  phosphate        A  6 

0.0256 

0.0032 

Av. 

0.0253 

0.0153 

0.0191 

0.0032 

-0.0010 

Timothy:              Bi 

0.0099 

0.0006 

Aq.-HCl-phenol         Hi' 

0.0099 

0.0005 

extraction              K3 

0.0096 

0.00291 

Av. 

0.0098 

0.0437 

0.0005 

-0.0011 

Timothy:              B4 

0.0249 

0.0029 

Aq.-HCl-phenol  extr.   Bs 

0.0246 

0.0030 

plus  phosphate         Bo 

0.0252 

0.00061 

Av. 

0.0249 

0.0153 

0.0151 

0.0029 

—0.0012 

Rice  Polish:            Ai 

0.0186 

0.0026 

Aq.-HCl  extraction     A2 

0.0192 

0.0032 

plus  phosphate        A3 

0.0182 

0.0034 

Av. 

0.0187 

0.0111 

0.0089 

0.0031 

0.0000 

A4 

0.0098 

0.0012 

Rice  Polish:           As 

0.0096 

• 

0.0012 

Aq.-HCl                A6 

0.0098 

0.0012 

extraction             A  7 

0.0098 

0.0012 

Av. 

0.0098 

0.0434 

0.0012 

-0.0004 

Rice  Polish:            Bi 

0.0150 

0.0017 

Aq.-HCl-phenol  extr.   B2 

0.0144 

0.0018 

plus  phosphate         B3 

0.0150 

Av. 

0.0148 

0.0111 

0.0110 

0.0017 

-0.0008 

B4 

0.0036 

Rice  Polish:            B5 

0.0040 

0.0014 

+0.0007 

Aq.-HCl-phenol        Be 

0.0040 

extraction             B7 

0.0036 

Av. 

0.0038 

0.0169 

iNot  included  in  average. 


10 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


TABLE  IV:     TEST  OF  COMPLETENESS  OF  EXTRACTION  AND  INFLU- 
ENCE OF  PHENOL  IN  THE  DETERMINATION  OF  INORGANIC 
PHOSPHORUS  IN  VEGETABLE  SUBSTANCES 


Treatment  and  sample  number 

First  extraction 

Second  extraction 

Sample 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Inor- 
ganic 
phos- 
phorus 

Percent 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Kxcess 
phos- 
phorus 
extracted 

(magne- 
sium 
pyro- 
phos- 
phate) 

Grams 

Gluten 
feed 

A q.-HCl  extraction                                   Ai 
Aq.-HCl  extraction                                   A2 
Aq.-HCl  extraction                                   A3 

Av. 
A q.-HCl-phenol  extraction                      Bi 
Aq.-HCl-phenol  extraction                     Ba 
A  q.-HCl-phenol  extraction                     Bs 

Av. 

0.0211 
0.0210 
0.0209 
0.0210 
0.0212 
0.0207 
0.0200 
0.0206 

0.0936 
0.0919 

0.0032 
0.0032 

0.0032 
0.0011 
0.0009 
0.0011 
0.0010 

0.0003 
-0.0024 

Brewer's 
grains 

Aq.-HCl  extraction                                   Ai 
A  q.-HCl  extraction                                  A2 
Aq.-HCl  extraction                                  A3 

Av. 
Aq.-HCl-phenol  extraction                      Bi 
Aq.-HCl-phenol  extraction                     B2 
Aq-HCl-phenol  extraction                    Bs 

Av. 

0.0026 

0.0028 

0.0028 

0.0027 

0.00121 

0.0022 

0.0026 

0.0024 

0.0120 
0.0107 

0.0010 
0.0010 
O.flOlO 
0.0010 
0.0010 
0.0010 
0.0010 
0.0010 

J.  0007 

- 

0.0006 

Timothy 

Aq.-HCl  extraction                                    Al 
Aq.-HCl  extraction                                   A2 
Aq.-HCl  extraction                                  A3 

Av. 
Aq.-HCl-phenol  extraction                     Bi 
Aq.-HCl-phenol  extraction                       B2 
Aq.-HCl-phenol  extraction                      B3 

Av. 

0.0062 
0.0040 
0.0025 
0.0042 
0.0105 
0.0097 
0.0106 
0.0103 

0.0187 
0.0459 

0.0019 

0.0016 
0.0018 
0.0017 
0.0003 
0.0006 

0.0004 

J.  0011 
-0.0010 

Wheat 

Aq.-HCl  extraction                                    Ai 
Aq.-HCl  extraction                                   A2 
Aq.-HCl  extraction                                  A3 

Av. 
Aq.-HCl-phenol  extraction                     Bi 
Aq.-HCl-phenol  extraction                     B2 
Aq.-HCl-phenol  extraction                     Bs 

Av. 

0.0092 
0.0096 
0.0092 
0.0093 
0.0040 
0.0049 
0.0048 
0.0046 

0.0415 
0.0205 

Wheat 
bran 

Aq.-HCl  extraction                                  Ai 
Aq.-HCl  extraction                                  A 2 
Aq.-HCl  extraction                                A3 

Av. 
Aq.-HCl-phenol  extraction                     Bi 
Aq.-HCl-phenol  extraction                    B2 
Aq.-HCl-phenol  extraction                     Bs 

Av. 

0.0143 
0.0134 
0.0138 
0.0138 
0.0145 
0.0157 
0.0140 
0.0147 

0.0615 
0.0655 

iNot  included  in  average. 


INORGANIC  PHOSPHORUS  ESTIMATION  11 

The  recovery  of  added  phosphorus  was  usually  incomplete,  the 
method  proving  unsatisfactory,  as  judged  by  this  measure.  The 
recovery  varied  from  a  minus  quantity  in  one  case,  with  blue  grass 
(Table  II,  p.  8)  to  100  percent  in  one  case,  with  rice  polish  (Table 
III,  p.  9).  The  variability  of  results  with  the  same  products,  in 
the  different  sets  of  estimations,  when  considered  in  connection  with 
the  difficulties  experienced  in  filtration,  suggested  to  us  that  at 
least  a  part  of  the  incompleteness  of  recovery  of  added  phosphates 
was  due  to  the  gelatinous  character  of  the  magnesia  mixture  pre- 
cipitate, notably  so  in  the  case  of  alfalfa,  as  shown  in  Tables  I  and 
II,  pp.  7  and  8.  In  the  work  reported  in  Table  II  the  precipitates 
remained  an  extra  day  in  acid-alcohol,  and  the  recovery  of  added 
phosphate  was  more  nearly  complete  than  in  the  work  reported  in 
Table  I.  A  further  consideration  of  this  factor  is  reported  in  Tables 
VIII  to  X  on  pp.  18  to  20. 

The  presence  of  phenol  gave  lower  results  for  inorganic  phos- 
phorus with  alfalfa,  brewer's  grains,  rice  polish,  gluten  feed  and 
wheat ;  and  higher  results  with  blue  grass,  timothy  and  wheat  bran. 
The  recovery  of  added  phosphate  was  usually  incomplete;  it  was 
almost  always  higher  with  phenol  than  without,  and  was  practically 
complete  with  brewer's  grains  (Table  I,  p.  7),  timothy  hay  and  rice 
poHsh  (Table  III,  p.  9).  With  timothy  hay  the  percent  of  inor- 
ganic phosphate  found  with  phenol  was  higher  than  without ;  the 
recovery  of  added  phosphate  was  complete  with  phenol,  and  appar- 
ently more  than  complete  without  phenol. 

It  is  not  clear,  from  these  results,  w^hat  is  the  nature  of  the 
effect  of  phenol  in  this  estimation.  Since  the  effect  of  phenol  is 
usually  to  lower  the  inorganic  phosphorus,  though  sometimes  to 
increase  the  same,  we  might  suppose  that  in  the  former  cases  cleav- 
age predominated,  except  as  suppressed  by  phenol,  while  in  the 
latter  cases  the  inhibited  processes  were  in  the  direction  of  syn- 
thesis. We  have  found  phenol,  as  used  in  this  work,  to  be  without 
effect  on  the  precipitation  of  magnesium  ammonium  phosphate 
from  pure  solutions.  Its  efficiency  to  prevent  enzymatic  cleavage 
was  not  experimentally  demonstrated. 

The  test  of  completeness  of  extraction  of  inorganic  phosphates 
by  0.2  percent  hydrochloric  acid  in  three  hours  was  made  with, 
gluten  feed,  brewer's  grains,  timothy  hay,  and  rice  polish,  the  re- 
sults being  given  in  Tables  III  and  IV  on  pp.  9  and  10.  In  con- 
sidering the  significance  of  the  weight  of  pyrophosphate  obtained 
from  the  second  extraction  one  should  bear  in  mind  the  fact  that 
this  is  due  largely  to  dissolved  phosphate  from  the  first  extraction 


12  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

remaining  adherent  to  the  foodstuff.  After  making  the  necessary 
correction  of  this  weight  by  subtracting  such  amount  of  pyrophos- 
phate as  corresponds  to  the  inorganic  phosphorus  in  the  liquid 
retained  by  the  sample,  the  results  are  very  small,  and  are  more 
often  minus  quantities  than  not,  showing  that  with  these  four  food- 
stuffs the  3-hour  extraction  is  practically  complete.  Further  tests 
of  the  completeness  of  the  3-hour  extraction  are  reported  in  Table 
VI,  p.  16. 

TEST  OF  THE  METHOD  OF  R.  C.  COLLISON 

As  a  possible  improvement  upon  the  method  thus  far  con- 
sidered the  similar  procedure  of  R.  C.  Collison*  was  studied.  Colli- 
son's  method  depends,  as  does  that  of  Forbes  and  associates,  from 
which  it  was  derived,  on  an  acid-alcohol  separation  of  inorganic 
phosphorus  from  phytin,  but  this  separation  takes  the  form  of  a 
direct  acid-alcohol  extraction  of  the  substance  to  be  analyzed.  The 
two  methods  differ  in  certain  other  details.  Coliison's  method  has 
the  advantage  of  being  much  more  easily  workable.  Unfortunately, 
however,  as  first  noted  by  Grindley  and  Ross,  and  later  substan- 
tiated in  this  laboratory,  results  from  the  use  of  this  method  are 
unsatisfactory  in  that  the  3-hour  extraction  is  either  insufficient 
or  causes  a  cleavage  of  inorganic  from  organic  phosphorus  com- 
pounds. A  second  and  even  a  third  3-hour  extraction  with  acid- 
alcohol  yields  considerable  amounts  of  inorganic  phosphorus. 

The  details  of  the  method  as  used  are  as  follows : 

METHOD  OF  R.  C.  COLLISON  FOR  THE  DETERMINATION  OF 
INORGANIC  PHOSPHORUS  IN  VEGETABLE  SUBSTANCES 

Weigh  out  10-gram  portions  of  the  samples  in  triplicate,  and  place  in  400 
c.  c.  Florence  flasks,  to  which  add  exactly  300  c.  c.  of  94-96  percent  phosphorus- 
free  alcohol,  containing  0.2  percent  of  hydrochloric  acid  (0.2  percent  actual 
HCl),  and  close  with  rubber  stopper. 

Shake  the  flasks  at  intervals  of  5  minutes  for  3  hours,  and  filter  through 
dry  double  filters  into  dry  flasks. 

Measure  out  250  c.  c.  aliquots  of  the  filtrates  into  400  c.  c.  beakers;  make 
just  alkaline  to  litmus  with  ammonia,  and  allow  to  stand  for  8-12  hours,  or 
over  night. 

Filter  through  double  filters,  and  wash  with  slightly  ammoniacal  94-96 
percent  alcohol.  In  case  a  small  portion  of  the  precipitate  resists  transfer 
from  the  beaker  by  the  usual  means  the  last  traces  may  be  dissolved  in  5  drops 
of  hydrochloric  acid,  with  the  assistance  of  a  rubber-capped  rod.  To  this  acid 
solution  add  10  c.  c.  of  alcohol;  make  slightly  alkaline  with  ammonia,  and  then 
transfer  to  the  filter. 

*Jour.  Ind.   and  Eng.   Chem.,   TV,  p.   606,    1912. 


INORGANIC  PHOSPHORUS  ESTIMATION  13 

Wash  several  times  with  ammoniacal  alcohol;  then  spread  out  the  inner 
papers  with  the  precipitate  and  allow  to  dry  completely.  Transfer  papers  and 
precipitates  to  Erlenmeyer  flasks  containing  exactly  100  c.  c.  of  0.5  percent 
aqueous  solution  of  nitric  acid  (0.5  percent  actual  HNO3).  Close  the  flasks  with 
rubber  stoppers;  shake  until  the  precipitates  are  thoroughly  broken  up,  and  let 
stand  over  night. 

Filter  through  dry  double  filters  into  dry  beakers;  pipette  out  75  c.  c.  of 
each  filtrate  and  determine  phosphorus  in  the  usual  way,  precipitating  first 
with  acid  molybdate  solution,  then  with  magnesia  mixture,  and  weigh  as  the 
pyrophosphate. 

If  the  final  solutions  are  highly  colored,  dissolve  the  pyrophosphates  and 
reprecipitate. 

Analytical  data  from  our  test  of  Collison's  method  are  set 
forth  in  the  following  table : 


14 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


< 

o 

o 
o 

t— I 


H 
H  CO 


O  O 


Q 
O 
» 
H 

O 

H 

» 


•d 

0 

i! 

V 

t~(»(M               c^q 

»-HO0t^                          O^ 

COLOC^J                     CO 

.-Hinm              o 

c 

"S, 

tointo              <o 

■-ICT>'*                     rt 

Ln--tto               in 

T-HCMCvi               eg 

rt 

o^o              o 

ooo              o 

t^ 

u 

a 

ooo              S 

OOO                   O 

O'O'O                   -o 

OOO                     O 

X 

0.) 

ooo             o 

odd             d 

ddd              d 

ddd             d 

3 

Ah 

o 

O 

'j-j 

ft 

u 

m 

O 
Si 
a, 

.2  o  u 

tfi 

LDCC-^CO        CO 

mcMcotr^to-^ 

coo'^c-q      in 

in  woo-*  ooo 

li 

u;  ^  +j 

s 

C^l  Cs]  CO  CO  CO  CO 

V 

!»  &  d 

oooo      o 

oooood 

oooo     o 

goooog 

•d 

o 

ss-a 

rt 

oooo      o 

oooooo 

oooo     o 

OOOCDOO 

o 

1) 

O 

oooo      o 

d  d>  d  d  <~> '^ 

dddd     d 

dddddd 

a 

|S 

m 

^  i 

.Sou 

Cfi 

CD  -^  ir>  oj  cvi  CO 

Ot^oo^^ooo 

OOOCV300      c^q 

O  CM  CM  ^  Ol  o 

05    J3    -w 

E 

CV5  C^J  CVI  -^  -^3*  ■<* 

■^-T'^  to  to  to 

<u    q.    rt 

ooooo  o 

oooo      o 

oooooo 

C    o   J3 

2 

oooooo 
oooooo 

oooooo 

o  o_  o  o      q 
ddod      o 

oooooo 
dddddd 

-3 
1) 

OJ 

■*-» 

> 

o 

c^ 

.-( 

q 

e-j 

'J 

O 

in 

d 

^ 

to 

£ 

u 

Ol 

CTV 

Ol 

00 

3 
u 
O 

ft 

CL( 

S 

6  ,'„ 

.5|  „ 

0) 

O          Nr-J 

■"j-to^in 

<OOCMO> 

t^^OlO 

tn  ^   *J 

E 

0|^T-|0 

OiOOlO 

•d 
11 

men! 
C    p  XI 

o      oo 

oooo 

oqqq 

oooo 

•d 
■a 

Sf    fc    » 

o'     od 

dodd 

dddo 

dddd 

5    |i. 

CB 

to  too 

tou^to 

to  to  to 

to  to  to 

E 

ooo 

ooo 

ooq 

ooo 

, 

O 

odd 

odd 

doo 

ddd 

P 

M   S 

4-) 

§   S 

c 

■^coo              <yi 

T-(tO<— 1                     CO 

O^-*                     CO 

CMinm              1-1 

i 

OlOm                     O) 

^oo              o 

oit^t^               oo 

II 

■^  •^•cf              -^ 

CMCMCM                   oq 

COCOCO                     CO 

ooo             o 

ooo             o 

qoq               q 

ooo             o 

ID 

ddd              d 

ddd              d 

ddd              d 

dod              o 

e  J, 

■B     O     V 

in 

tOOOlOtO        00 

O-— *0^t0-«j' 

t^oooo-^ooo 

00 -rt '^  CM  en -<# 

00  00  00  oooooo 

m   S3    ii 

B 

CDtDCDt^        t~ 

OlCIlCTlOOO 

-ct  ^  M- in  in  to 

a;    p,   rt 

ooo*— <t— I^H 

F    P  X! 

OOOO        O 

oooooo 

OOOOOO 

oooooo 

he   ^    0. 

O 

<:i<:><dd     d 

dddddd 

dddddd 

(Z>ddddd 

S    ^ 

i-H  cv)  CO  ^  Ln  to  M 

^  CM  CO'*  in  to  01 

<—<  CM  CO -^  in  to  01 

T-*  CM  CO  ■<*  in  to  01 

1) 

u 

<u 

0) 

ho 

ho 

ho 

h( 

<« 

rt 

rs 

ft 

t-i 

u 

u 

u 

H 

jj 

V 

(U 

> 

> 

> 

< 

^ 

< 

-< 

« 

a  V  '0 

»   3J   4) 

o  13  <a 

D  D  4J 

0. 

a 

*->  -i-t  *j 

a  n  ^ 

rt  c3  rt 

rt  rt  rt 

rt  rt  (« 

XIJ3X1 

Xi^  J= 

s:s:si 

xijaxi 

rt 

D.  ?.  p. 

a  D.  0. 

ft  O.  ft 

ft  ft  ft 

Cfi 

a.  1/1  01 

01    01    0) 

01    0)    01 

t/i  m  in 

ooo 

coo 

ooo 

OOO 

j:^:^: 

-  -  --c-s^ 

sisisi 

J3Xlja 

a  o.  P. 

£  S  £  D.  a  G 

ftft  a 

C  C  C  ft  ft  ft 

C  C  C  01  01  01 

t.    U,    U    [-,    u)    01 
4)    U    (U    3    3    3 

s:  si  si  111  in  m 
.£.2.2  3  3  3 

!*    i'    ^   (B    01    01 

I;     I,  4)  0)  u 

rt  rt  rt  oj  «  0) 

OOO   ft  ft  ft 
ft  ft  P*  D  (U   JJ 

+j  .p  4J    ft  ft  ft 

«  rt  rt  oj  u  J) 

eeeEEE 

^^^EEE 

S  8  S  1  E  S 

^^^SES 

o  o  o  rt  rt  rt 

^^^c^c2c2 

kSkccckcc 

j>  K^  [->  ra  ra  flS 

INORGANIC  PHOSPHORUS  ESTIMATION  15 

FURTHER  CONSIDERATION  OF  THE  METHOD  OF 
FORBES  AND  ASSOCIATES 

Since  Collison's  method  proved  unsatisfactory,  further  work 
was  done,  at  a  later  date,  in  attempts  to  improve  upon  the  method  of 
Forbes  and  associates  (p.  5).  In  this  work  we  gave  attention  to 
the  following  points: 

(a)  The  completeness  of  extraction,  (b)  the  effect  of  using 
much  larger  amounts  of  magnesia  mixture  in  the  precipitation,  (c) 
the  allowing  of  more  time  for  the  precipitation  with  magnesia  mix- 
ture, (d)  the  facilitating  of  filtration  by  the  use  of  the  centrifuge, 
and  (e)  the  use  of  mechanical  means  to  break  up  the  precipitate  in 
acid-alcohol  to  insure  the  complete  solution  of  the  phosphate. 

The  following  tabular  data  set  forth  the  results  of  this  study, 
the  general  method  being  that  of  the  recovery  of  known  amounts 
of  phosphate  introduced  into  the  estimation.  The  details  were  as 
specified  on  p.  5,  though  phenol  was  used  only  in  the  tests  reported 
in  Table  IX,  p.  19.  Throughout  this  work  the  centrifuge  was  used 
to  facilitate  filtration  of  the  0.2  percent  hydrochloric  acid  extracts. 
The  advantage  derived  from  this  treatment  was  very  great. 
Extracts  which  it  was  impossible  otherwise  to  filter  within  a  reason- 
able time  were,  with  the  aid  of  the  centrifuge,  filtered  without 
difficulty  or  delay. 

Other  constant  conditions  in  this  work  were  the  use  of  an 
extreme  amount  (50  c.  c.)  of  magnesia  mixture  in  the  preliminary 
precipitation  (instead  of  10  c.  c.  as  usual) ;  and  three  days'  time  were 
allowed  in  all  cases  for  the  completion  of  this  precipitation. 

Table  VI,  p. 16,  reports  results  from  a  test  of  the  Acid-Alcohol 
Method  of  Forbes  and  associates  (for  details  see  p.  5;  modified  as 
above),  with  alfalfa  hay,  by  the  method  of  recovery  of  added  phos- 
phates, and  a  test  of  the  completeness  of  extraction.  Alfalfa  was 
selected  for  this  test  as  that  substance  which,  in  our  previous 
experience,  had  given  us  the  most  trouble  and  the  poorest  results. 
Samples  4,  5  and  6  as  compared  with  1,  2  and  3,  show  that  the 
recovery  of  added  phosphates  was  incomplete,  except  in  the  case 
of  sample  5,  in  which  case,  as  explained  in  the  footnote  below  the 
table,  on  account  of  the  accidental  breaking  of  the  first  filter  paper, 
the  precipitation  was  finally  made  in  the  presence  of  the  pulp  from 
this  paper.  In  this  case  the  recovery  was  complete.  This  acci- 
dental result  sustained  our  hypothesis  that  our  difficulty  in  recover- 
ing added  phosphates  was  due  to  the  physical  character  of  the  first 
magnesia  mixture  precipitate,  its  gummy  character  rendering  im^- 
possible  the  complete  separation,  in  acid-alcohol,  of  the  inorganic 
phosphates  from  the  phytin  and  other  substances  present.  This 
point  was  given  further  study. 


16 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


Determinations  7a,  8a  and  9a  were  made  in  the  same  way  as 
1,  2  and  3.  Determinations  7b,  8b  and  9b  were  second  extractions 
of  the  residues  from  determinations  7a,  8a  and  9a.  The  results 
from  the  second  extraction  equalled  only  the  residual  amount  of 
phosphate  clinging  to  the  sample,  from  the  iirst  extraction ;  that  is, 
no  more  inorganic  phosphate  was  dissolved  in  a  three-hour  extrac- 
tion, following  the  three-hour  extraction  regularly  prescribed  in  the 
method.  The  extraction  was  complete  at  the  end  of  the  first  three- 
hour  treatment. 


TABLE  VI:     TEST  OF  INORGANIC  PHOSPHORUS  ESTIMATION 

ON  VEGETABLE  SUBSTANCES  BY  THE  ACID-ALCOHOL 

METHOD  (WITHOUT  PHENOL) 


Sample 
No. 

Treatment 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Percent 
inor- 
ganic 
phos- 
phorus 

Added 
phos- 
phorus 
(magne- 
sium 
pyro- 
phos- 
phate) 

Grams 

Added  phos- 
phorus recovered 

Sub- 
stance 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Percent 

Alfalfa 
hay 

1 
2 
3 

Filtrate  precip.  with  50c.  c. 

magnesia  mixture +25 c.  c. 

ammonia;  without  added 

phosphate 

.0176 
.0181 
.0178 

Average 

.0178 

.0992 

Alfalfa 
hay 

4 

5* 

6 

Same,  with  added 
phosphate 

.0425 
.0477 
.0404 

.0299 
.0299 
.0299 

.0246 
.0299 
.0226 

82.27 
100.00 
75.58 

Average 

.0435 

85.95 

Alfalfa 
hay 

7a 
8a 
9a 

Without  added  phosphate; 
same  as  1,  2  and  3 

.0182 
.0181 
.0181 

Average 

.0181 

.1008 

Alfalfa 
hay 

7b 

8b** 

9b 

Second  extraction  of 
samples  7a,  8a  and  9a 

.0062 
.0060 
.0058 

.0060*** 

.0060 

.0060 

Average 

.0060 

Weight  of  samples  10  grams;  results  represent  one-half  of  this  amount. 

*During  the  filtration  of  the  first  magnesia  mixture  precipitate  the  filter -paper  broke. 
This  paper  was  then  added  to  the  beaker  containing  the  precipitate,  was  beaten  up  into  a  pulp, 
and  the  filtration  continued  through  a  new  paper. 

** Samples  7b,  8b  end  9b  are  the  extraction  residues  from  7a,  8a  and  9a,  with  filter  paper 
added,  and  also  enough  0.2  percent  hydrochloric  acid  solution  to  make  the  original  volume 
of  300  c.  c.  This  set  was  extracted  for  three  hours  to  test  the  completeness  of  the  previous 
extraction. 

Magnesium  pyrophosphate  equivalent  to  phosphorus  in  solution,  from  previous  extrac* 
tion,  remaining  in  the  sample. 


INORGANIC  PHOSPHORUS  ESTIMATION 


17 


Considering  the  data  in  Table  VII,  below,  with  samples  1-9  the 
first  magnesia  mixture  precipitates  were  extracted  with  200  c.  c.  of 
0.2  percent  nitric  acid  in  alcohol  instead  of  100  c.  c.  as  usual,  in 
order  to  test  the  sufficiency  of  the  latter  amount  to  neutralize  the 
ammonia  remaining  in  the  precipitates,  and  to  dissolve  the  phos- 
phates. With  samples  10-12  the  usual  100  c.  c.  of  acid  alcohol  were 
used.  The  comparison  shows  that  100  c.  c.  of  acid  was  probably 
sufficient,  though  the  result  from  sample  11,  for  some  unknown 
reason  was  low. 

The  second  and  third  sets  of  triplicates,  samples  4-9,  contrast 
results  from  the  addition  of  phosphates  (to  be  recovered)  after  the 
extraction  (immediately  before  precipitation)  and  previous  to  the 
three-hour  extraction.  We  see  here  no  evidence  of  a  retention  of 
added  phosphates  by  the  solid  substance  of  the  sample. 


TABLE  VII:     TEST  OF  INORGANIC  PHOSPHORUS  ESTIMATION 

ON  VEGETABLE  SUBSTANCES  BY  THE  ACID-ALCOHOL 

METHOD   (WITHOUT  PHENOL) 


Sample 
No. 

Treatment 

Mag- 
nesium 
pyro- 
phos- 
phate 

Gm. 

Added 
phos- 
phorus 
(mag- 
nesium 
pyrophos- 
phate) 

Gm. 

Added  phosphorus 
recovered 

Substance 

Mag- 
nesium 
pyrophos- 
phate 

Gm. 

Percent 

Alfalfa 
hay 

1 
2 
3 

Ave. 

Filtrate  precipitated   with   50  c.   c. 
mag.  mixture  and  20  c.  c.  ammonia, 
200  c.  c.  acid  alcohol  used  to  extract 
magnesia  mixture  precipitate. 

.0183 
.0180 
.0184 

.0182 

.0000 

Alfalfa 
hay 

4 
5 
6 

Ave. 

Same  as  above  +  25  c.  c.  phosphate 
solution  just  before  precipitation. 

.0603 
.0606 
.0604 

.0604 

.04485 

.0429 

94.1 

Alfalfa 
hay 

7 
8 
9 

Ave. 

Same  as  1.  2  and  3  except  that  25  c.c. 
phosphate  solution  added,  shaken  for 
3  hrs.,   filtered  and  filtrate  precip. 
as  1,  2  and  3. 

.0466 
.0466 
.0472 

.0299           .0284 ) 
.0284  f 
.0290 

94.98 
96.99 

95.98 

Alfalfa 
hay 

10 
11 
12 

Ave. 

Same  as  7,  8  and  9,  except  that  only 
100  c.  c.    acid   alcohol  were  used    in 
extraction  of  magnesia  mixture  pre- 
cipitate. 

.0467 
.0447 
.0464 

.0285 
.0265 
.0282 

95.32 
88.63 
94.31 

92.75 

Alfalfa 
bay 

13 
14 
15 

Ave. 

25  c.   c.   phosphate  solution   put   in 
flasks-|-175  c.  c.  .2%  nitric  acid  in  alco- 
hol+2  filter  papers,  shaken,  filtered 
and  aliquot  taken  for  precipitation. 

.0404 
.0404 
.0402 

.03987 

.0404 
.0404 
.0402 

101.1 

Weight  of  samples,  10  grams;  results  represent  0.5  of  this  amount. 


18 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


Determinations  13-15  were  made  to  ascertain  whether  or  not 
we  could  get  a  complete  recovery  of  phosphates  from  filter  paper 
pulp.  It  was  possible  completely  to  recover  the  phosphates.  This 
test  has  a  bearing  on  work  to  follow,  and  shows  that  the  incomplete 
recovery  of  added  phosphates  could  not  be  due  to  the  presence  of 
filter  paper  pulp. 

The  recovery  of  added  phosphates  in  these  estimations  on 
alfalfa  was  fairly  satisfactory. 


TABLE  VIII :     TEST  OF  INORGANIC  PHOSPHORUS  ESTIMATION  ON 

VEGETABLE  SUBSTANCES  BY  THE  ACID-ALCOHOL 

METHOD  (WITHOUT  PHENOL) 


Sample 
No. 

Treatment 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Added 
phos- 

Added phos- 
phorus recovered 

Sub- 
stance 

Percent 
inor- 
ganic 
phos- 
phorus 

phorus 
(magne- 
sium 
pyro- 
phos-  ■ 

phate) 

Grams 

Magne- 
sium 
pyro- 
phos-., 
phate 

Grams 

Percent 

Blue 

grass 

■     1 
2 
3 

Average 

Filtrate  precip.  with  50  c.  c- 
magnesia  mixture -^25  c- c. 
ammonia,  plus  paper  pulp 

.0373 
.0371 
.0386 

.2078 
.2067 
.2151 

.2099 

Blue 
grass 

4 
5 
6 

Average 

Same  +25  c.  c.  phosphate 
solution 

.0624 
.0623 
.0633 

.0250 
.0250 
.0250 

.0251 
.0252 
.0247 

100.04 
100.08 
98.80 

99.97 

Rice 
polish 

7 
8 
9 

Average 

Same  as  1,  2  and  3 

.0070 
.0070 

.0070 

i0390 

Rice 
polish. 

10 
11 
12 

Average 

Same+25c.  c  phosphate 
solution 

.0295 
.0295 

.0250 
.0250 

.0225 
.0225 
.0225 

90.00 

Weight  of  samples  10  grams;  results  represent  0.5  of  this  amount 


INORGANIC  PHOSPHORUS  ESTIMATION 


19 


table  ix:   test  of  inorganic  phosphorus  estimation  on 

vp:getable  substances  by  the  acid-alcohol 

method  (with  phenol) 


Sam- 
ple 
No. 

Treatment 

Mag- 
nesium 
pyro- 
phos- 
phate 

Gm. 

Percent 
inor- 
ganic 
phos- 
phorus 

Added 

phos- 
phorus 
(mag- 
nesium 
pyro- 
phos- 
phate) 
Gm. 

Added  phos- 
phorus recovered 

Substance 

Mag- 
nes  i  u  m 
pyro- 
phos- 
phate 
Gm. 

Per- 
cent 

Rice  polish 

1 
2 
3 

Ave. 

Extracted  with  0.2%  hydrochlo- 
ric acid  +  50  u m.  phenol  per  liter: 
filtrate    precipitated   with  mag- 
nesia mixture  +  25  c.c.  ammonia. 

.0080 
.0072 

.0446 
.0401 

.0424 

Rice 

polish  + 

phosphate 

4 
5 
6 

Ave. 

Same 

.0543 
.0461 
.0443 

.0768 
.0768 
.0768 

.0467 
.0385 
.0367 

60.81 
50.13 
47.79 

52.91 

Middlings 

7 
8 
9 

Ave. 

Same 

.0132 
.0133 
.0129 

.0131 

.0724 

Middling-s 
-f  phos- 
phate 

10 
11 
12 

Ave. 

Same 

.0830 
.0864 
.0847 

.0768 
.0768 
.0768 

.0699 
.0733 
.0716 

91.01 
95.44 
93.23 

93.23 

Soy  beans 

13 

14 
15 

Ave. 

Same 

.0102 
.0105 
.0102 

.0103 

.0574 

Soy  beans+ 
phosphate 

16 

17 
18 

Ave. 

Same 

.0790 
.0801 
.0823 

.0805 

.0768 
.0768 
.0768 

.0687 
.0698 
.0720 

89.45 
90.89 
93.75 

91.36 

Weight  of  samples  10  grams;  results  represent  0.5  of  this  amount. 


20 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


TABLE  X:     TEST  OF  INORGANIC  PHOSPHORUS  ESTIMATION  ON 

VEGETABLE  SUBSTANCES  BY  THE  ACID-ALCOHOL 

METHOD  (WITHOUT  PHENOL) 


Sub- 
stance 

Sample 
No. 

Treatment 

Magne- 
sium 
pyro- 
phos- 
phate 

Gram 

Percent 
inor- 
ganic 
phos- 
phorus 

Added 
phos- 
phorus 
(magne- 
sium 
pyro- 
phos- 
phate) 

Gram 

Added  phos- 
phorus recovered 

Magne- 
sium 
pyro- 

phos-        Percent 
phate 

Gram 

Soy 
beans 

1 
2 
3 

Average 

Usual  method,  plus  iilter 

paper  pulp;\vithout 

phosphate 

.0102 
.0103 
.0101 

.0102 

.0568 

Soy 
beans 

1 
2 
3 

Average 

Same,  plus  phosphate 

.0336 
.0345 
.0347 

.0261 
.0261 
.0261 

.0234 
.0243 
.0245 

90.03 
93.10 
93.87 

92.33 

Mid- 
dlings 

1 
2 
3 

Average 

Same,  without  phosphate 

.0135 
.0131 
.0139 

.0135 

.0752 

Mid- 
dlings 

1 
2 
3 

Average 

Same,  with  phosphate 

.0432 
.0430 
.0418 

.0261 
.0261 
.0261 

,0297 
.0295 
.0283 

113.79 
113.02 
108.42 

111.74 

Oat 

straw 

1 
2 
3 

A  verage 

Same,  without  phosphate 

.0062 
.0058 
.0061 

.0060 

.0334 

Oat 
straw 

1 
2 
3 

Average 

Same,  with  phosphate 

.0312 
.0319 
.0318 

.0261 
.0261 
.0261 

.0252 
.0259 
.0258 

96.55 
99.23 
98.85 

98.21 

Weight  of  samples  10  grams;  results  represent  0.5  of  this  amount. 


Table  VIII,  p.  18,  sets  forth  results  of  estimations  on  blue  grass 
and  rice  polish,  with  and  without  added  phosphate,  with  filter  paper 
pulp  added,  in  the  first  precipitation,  to  maintain  a  readily  permeable 
condition  in  the  precipitate. 

With  blue  grass  the  results  may  be  considered  perfect.  With 
rice  polish  the  recovery  of  added  phosphates  was  90  percent  efficient. 
The  loss  amounted  to  0.0025  gm.  magnesium  pyrophosphate  pel* 
determination. 


INORGANIC  PHOSPHORUS  ESTIMATION  21 

Table  IX,  p.  19,  is  a  record  of  determinations  on  rice  polish, 
wheat  middlings  and  soy  beans,  with  and  without  added  phosphate, 
with  phenol  added  to  the  extractive  reagent  to  prevent  possible 
enzyme  action  involving  phosphorus  compounds,  and  with  filter 
paper  pulp  added  to  facilitate  solution  of  the  phosphates  in  the  pre- 
cipitate. The  recovery  of  added  phosphates  was  unsatisfactory; 
the  effect  of  the  phenol  on  the  physical  condition  of  the  first  mag- 
nesia mixture  precipitate  being  of  such  a  nature  as  to  hinder  the 
dissolving  out  of  the  included  phosphates. 

Table  X,  p.  20,  sets  forth  results  from  determinations  on  soy 
beans,  wheat  middlings  and  oat  straw,  with  and  without  added  phos- 
phate, with  filter  paper  pulp,  and  without  phenol  added  to  the  ex- 
tracting sample.  With  oat  straw  the  results  were  satisfactory. 
With  soy  beans  the  recovery  of  added  phosphates  was  incomplete, 
while  with  wheat  middlings  we  seem  to  have  recovered  3  milligrams 
more  phosphate  than  was  added.  In  this  case  we  can  not  ascribe 
the  imperfection  of  the  result,  as  usual,  to  the  physical  condition  of 
the  magnesia  mixture  precipitate.  Here,  it  seems,  that  there  must 
have  been  a  cleavage  of  inorganic  from  organic  phosphorus,  either 
enzymatic  or  as  a  result  of  the  extractive  treatment.  Anderson* 
has  shown  that  0.2  percent  hydrochloric  acid  is  not  sufficiently  con- 
centrated to  prevent  enzymatic  hydrolysis  of  the  phytin  of  wheat 
bran,  and,  since  middlings  contains  bran,  this  observation  must 
apply  to  middlings  as  well.  How  general  may  be  enzymatic 
hydrolysis  of  organic  phosphorus  compounds  in  our  0.2  percent 
hydrochloric  acid  extractive  reagent  we  are  unable  to  say.  In  the 
case  of  wheat  middlings  only  has  the  extent  of  the  hydrolysis  been 
shown,  in  our  work,  to  be  sufficient  more  than  to  offset  the  various 
factors  tending  to  give  low  results  for  inorganic  p^iosphorus.  Our 
second  three-hour  extractions  of  timothy,  rice  polish,  gluten  feed 
and  brewer's  grains  showed  no  considerable  additional  inorganic 
phosphorus  resulting  either  from  solution  or  hydrolytic  decompo- 
sition of  organic  compounds. 

CONCLUSIONS  FROM  WORK  ON  INORGANIC  PHOSPHORUS 
ESTIMATION  IN  VEGETABLE  SUBSTANCES 

(1)  A  three-hour  extraction  with  0.2  percent  hydrochloric  acid 
in  water  appears  to  accomplish  practically  complete  solution  of  the 
inorganic  phosphates  of  finely  ground  vegetable  substances,  but  in 
the  case  of  wheat  middlings  was  shown  to  allow  enzymatic  hydroly- 
sis of  organic  phosphorus,  with  the  liberation  of  inorganic  phos- 
phate. 

*Anderson,  R.  J.:    Journ.  Biol.  Chem.  XX  (1915)  483-491. 


22  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

(2)  The  introduction  of  filter  paper  pulp  into  such  an  extract 
materially  assists  in  the  maintenance  of  an  easily  penetrable  con- 
dition in  a  magnesia  mixture  precipitate  from  the  same. 

(3)  It  was  found  possible  completely  to  recover  phosphates 
from  filter  paper  pulp  alone  as  used  in  this  work. 

(4)  The  use  of  the  centrifuge  very  greatly  facilitates  the  fil- 
tration of  dilute  aqueous-acid  extracts  of  vegetable  substances. 

(5)  There  has  appeared  no  reason  to  doubt  the  completeness 
of  the  precipitation  of  the  inorganic  phosphates  from  the  0.2  percent 
hydrochloric  acid  solution,  through  the  use  of  magnesia  mixture 
and  ammonia. 

(6)  The  separation  of  the  inorganic  phosphates  from  the 
phytin  and  other  constituents  of  the  magnesia  mixture  precipitate, 
through  the  agency  of  0.2  percent  nitric  acid  in  95  percent  alcohol, 
is  attended  by  difficulties  which  have  not  yet  been  overcome.  That 
these  difficulties  are  largely  physical,  as  determined  by  the  bulky 
and  often  gummy  nature  of  the  magnesia  mixture  precipitate,  seems 
to  be  a  fact.  That  they  are  in  part  of  a  chemical  nature,  and  due 
to  the  cleavage  of  phytin  or  other  organic  phosphorus  compounds 
of  the  magnesia  mixture  precipitate  through  the  agency  of  enzymes 
appears  also  to  be  true. 

(7)  The  use  of  phenol  (50  gm.  per  liter)  in  the  extractive 
reagent  was  shown  not  to  affect  the  precipitation  and  estimation 
of  phosphates  in  pure  solutions.  In  the  estimation  of  inorganic 
phosphorus  in  extracts  of  vegetable  substances  the  presence  of 
phenol  appeared  to  favor  the  recovery  of  added  phosphates.  Phenol, 
when  used  in  this  way,  sometimes  increased  but  more  commonly 
decreased  the  inorganic  phosphorus.  In  extracts  of  certain  vege- 
table products  the  presence  of  phenol  increased  the  difficulty,  rather 
commonly  experienced,  in  breaking  up  the  magnesia  mixture  pre- 
cipitate in  acid  alcohol. 

(8)  Modification  of  the  acid-alcohol  method  of  Forbes  and 
associates  by  the  introduction  of  filter  paper  pulp  into  the  extract 
from  which  the  phosphates  are  to  be  precipitated,  the  use  of  exces- 
sive amounts  of  magnesia  mixture  in  this  first  precipitation,  and 
allowing  unusual  duration  of  time  for  this  precipitation  gave  appar- 
ently perfect  results,  as  judged  by  recovery  of  added  phosphates,  in 
certain  cases,  but  unsatisfactory  results  in  others. 

(9)  Incompleteness  of  recovery  of  added  phosphates  w^s  shown 
not  to  be  due  to  retention  of  phosphates  by  the  solid  substance  of 
the  sample. 


INORGANIC  PHOSPHORUS  ESTIMATION  23 

(10)  We  are  unable  to  recommend  this  method,  or  any  other,  as 
rehable  for  the  estimation  of  inorganic  phosphorus  in  vegetable 
substances  generally. 

(11)  The  acid-alcohol  extraction  of  the  method  of  R.  C.  Collison 
is  either  incomplete,  in  three  hours,  or  else  causes  a  cleavage  of 
organic  compounds  of  phosphorus,  with  the  liberation  of  inorganic 
phosphate. 

THE  ESTIMATION  OF  WATER-SOLUBLE  INORGANIC  PHOS- 
PHORUS IN  ANIMAL  SUBSTANCES 

The  method  of  determination  of  inorganic  phosphorus  in  animal 
substances,  as  published  from  this  laboratory,  is  an  adaptation  of 
the  usual  magnesia  mixture  method  for  phosphorus  estimation  to 
the  conditions  of  work  with  water-extracts  of  animal  products.  The 
original  points,  therefore,  in  our  procedures,  as  specified  for  various 
animal  tissues,  are  mechanical  and  chemical  details  of  extraction, 
filtration  and  precipitation.  Considerable  care  has  been  bestowed 
upon  the  quantitative  proof  of  the  correctness  of  these  details,  es- 
pecially as  providing  for  complete  extraction,  rapid  filtration,  and 
prevention  of  the  cleavage  of  the  organic  phosphorus  compounds 
from  which  the  inorganic  are  to  be  separated.  These  studies  are  in 
the  nature  of  comparisons  of  the  neutral  molybdate  method  of 
Emmett  and  Grindley,  the  barium  chlorid  method  of  Siegfried  and 
Singewald  and  the  magnesia  mixture  method  of  Forbes  and  asso- 
ciates, usually  checked  by  the  recovery  of  known  amounts  of  added 
phosphate ;  and  they  appear  to  result  in  the  establishment  of  the 
last-named  method  as  reliable  and  workable  with  a  wide  range  of 
animal  t:[ssues. 

In  the  use  of  this  method  One.  should  recognize  the  fact  that 
it  seeks  to  estimate  the  water-soluble  inorganic  phosphates  only. 
This  figure  would  be  at  least  practically  the  same  as  total  inorganic 
phosphates  for  most  animal  tissues  and  products,'  but  in  the  case 
of  tissues  such  as  bone,  w:hich  contain  large  amounts  of  inorganic 
phosphates  which  are  insoluble  in  w^ater,  this  method  is  inapplicable. 

,TJhe  work  on^animal  tissues  was  done  in  three  series  of  determi- 
nations, one  each  in  the  years  1912,  1913  and  1914.  The  remainder 
of  this  paper  sets  forth  the  details  of  this  work.  The  discussion 
of  th6  results  of  the  work  of  1912-1913  will  be  found  on  pages  33 
and  34,  and  of  19 U  on  pages  38  to  40. 

^^  riv-i-  or---:  f;T     .     THE  WORK  OF  1912 --■  ?~ 

The  work 'Of  1912  .consisted  of  a  comparison  of  th6  methods  of 
Emmett. an^  Grindley,  Siegfried  and  Singewald,  and  Forbes  and 
associates  with  muscle,  blood  and  brain.     The  muscle  used  was  a 


24 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


vacuum-dried  product  prepared  in  the  laboratory  of  Dr.  H.  S.  Grind- 
ley,  of  the  University  of  Illinois.  The  following  schedule  shows 
methods  of  precipitation,  amounts  of  extracts  used,  number  of  re- 
peats, and  amount  of  standard  phosphate  solution  added  to  certain 
of  these  determinations : 


A     Neutral  Molybdate 
Precipitation 

(Emmett  and  Grindley) 


A-1  500  c.  c.  extract 

A-2  500  c.  c.  extract 

A-3  250  c.  c.  extract  +  25  c.  c. 

phosphate  solution 
A-4  250  c.  c.  extract  +  25  c.  c. 

phosphate  solution 


1.  Muscle 

2.  Blood 

3.  Brain 


B-1  500  c.  c.  extract 
B-2  500  c.  c.  extract 
B-3  250  c.  c.  extract  +  25  c.  c. 

phosphate  solution 
(Siegfried  and  Singewald)    B-4  250  c.  c.  extract  +  25  c.  c. 

phosphate  solution 


B     Barium  Chlorid 
Precipitation 


Magnesia  Mixture 
Precipitation 

(Forbes,  et  al) 


C-1  500  c.  c.  extract 

C-2  500  c.  c.  extract 

C-3  250  c.  c.  extract  +  25  c.  c. 

phosphate  solution 
C-4  250  c.  c.  extract  +  25  c.  c. 

phosphate  solution 


The  work  with  muscle  was  performed  on  a  cold  water  extract 
prepared  as  specified  below: 

OUTLINE  FOR  THE  PREPARATION  OF  A  COLD  WATER  EXTRACT 

OF  DESICCATED  FLESH  FOR  THE  DETERMINATION 

OF  INORGANIC  PHOSPHORUS 

Weigh  out  about  45  grams  of  the  vacuum-dried  meat,  and  divide  it  among 
sixteen  150  c.  c.  beakers.  To  each  beaker  with  its  contents  add  about  3-5  c.  c. 
of  distilled  water.  Break  up  any  lumps  and  stir  well  with  a  glass  rod  until 
the  mass  forms  a  thick  paste.  Add  50  c.  c.  of  distilled  water  to  each  beaker 
and  stir  thoroughly  for  15  minutes.  Allow  the  insoluble  portion  to  settle  for 
a  few  minutes  (3-5)  and  decant  the  supernatant  liquid  through  wet  11  cm. 
filters.  Collect  the  filtrates  in  250  c.  c.  Florence  flasks.  Take  care  that  the 
funnels  touch  the  sides  of  the  necks  of  the  flasks.  Drain  the  residues  thor- 
oughly, keeping  as  much  of  them  in  the  beakers  as  possible.  Treat  these 
residues  with  25  c.  c.  of  distilled  water,  stirring  for  5-7  minutes,  and  then  allow- 
ing 3-5  minutes  for  the  solid  particles  to  settle  before  filtering.  Decant,  etc., 
as  described  above.  Repeat  this  last  treatment  until  the  filtrate  measures 
about  220  c.  c.  Then  transfer  the  entire  residue  to  the  filter  and  wash  twice 
with  about  8-10  c.  c.  of  distilled  water.  Allow  all  the  liquid  to  pass  through 
the  filter  before  adding  the  next  extract.  Whenever  the  major  portion  of  the 
residue  has  become  mechanically  transferred  to  the  filter,  return  it  to  the 
beaker,  using  great  care  not  to  break  the  filter  paper.  Take  the  sixteen  filtrates 
of  about  250  e,  c.  each  and  transfer  all  of  them  to  a  measuring  flask.  Wash  out 
each  Florence  flask  twice,  using  about  5-8  c.  c.  of  distilled  water  each  time. 
Make  the  extract  up  to  5000  c.  c.  and  mix  it  thoroughly  without  too  much 
mechanical  agitation. 


INORGANIC  PHOSPHORUS  ESTIMATION  25 

With  blood,  the  comparisons  were  made  on  an  extract  prepared 
with  the  aid  of  ammonium  sulphate,  the  details  of  procedure  being 
as  specified  below: 

METHOD  FOR  THE  PREPARATION  OF  HOT  WATER-AMMONIUM 
SULPHATE  EXTRACT  OF  BLOOD 

Weigh  about  50  grams  of  fresh  blood,  or  its  equivalent  of  oxalated  blood, 
into  each  of  six  400  c.  c.  beakers.  To  each  beaker  add  a  few  c.  c.  of  distilled 
water  and  work  up  the  blood  and  water  with  a  glass  rod.  Make  up  to  about 
200  c.  c.  with  boiling  distilled  water.  Place  over  a  flame  and  gradually  bring 
to  boiling,  with  constant  stirring.  When  boiling  begins  add  to  each  beaker 
20  c.  c.  of  20  percent  ammonium  sulphate  solution.  Boil  with  constant  stirring 
for  about  10  minutes.  Decant  onto  sand  on  linen.  When  the  liquid  is  through 
lift  the  coagulum  oif  from  the  sand  and  transfer  to  a  mortar.  Grind  the  coag- 
ulum  to  a  smooth  paste  and  transfer  from  mortar  to  beaker  with  boiling  dis- 
tilled water.  Make  up  to  about  80  c.  c.  with  the  same.  Stir  for  8  minutes  and 
pour  contents  again  onto  the  sand  filter.  After  the  extract  is  through  return 
the  coagulum  to  the  mortar  and  grind  a  second  time,  transferring  to  the  beaker 
as  before  with  boiling  distilled  water.  Repeat  this  process  of  8-minute  extrac- 
tions of  the  coagulum  in  hot  water  and  filtration  as  above  directed,  without 
further  grinding,  until  the  filtrates  measure  about  750  c.  c.  each.  Wash  out 
each  beaker  twice  with  8-10  c.  c.  hot  distilled  water,  completing  the  transfer  of 
the  coagulum  and  extract  to  the  sand.  Wash  the  coagulum  on  the  sand  twice 
with  boiling  water  from  a  wash  bottle.  At  all  times  allow  the  filter  to  drain 
well  between  additions  of  extract  or  wash  water.  Combine  the  six  filtrates  of 
about  800  c.  c.  each,  washing  out  the  containers  of  each  twice  with  distilled 
water.     Make  the  extract  up  to  5000  c.  c.  and  mix. 

Still  a  different  method  of  manipulation,  with  the  aid  of  am- 
monium sulphate,  was  necessary  for  the  extraction  of  brain.  The 
details  are  as  specified  below: 

DIRECTIONS  FOR  THE  PREPARATION  OF  HOT  WATER-AMMONIUM 
SULPHATE  EXTRACT  OF  BRAIN 

Weigh  out  about  10  grams  of  brain  into  each  of  ten  250  c.  c.  beakers.  To 
each  beaker  add  a  few  c.  c.  of  distilled  water  and  work  up  the  brain  and  water 
with  a  glass  rod.  Make  up  to  about  100  c.  c.  with  boiling  water.  Place  over 
a  flame  and  gradually  bring  to  boiling,  with  constant  stirring.  After  boiling 
has  begun  add  to  each  beaker  20  c.  c.  of  20  percent  ammonium  sulphate  solution. 
Boil  for  about  10  minutes.  Allow  to  settle  for  a  moment  and  decant  liquid  onto 
sand  on  linen.  In  case  the  extracts  do  not  filter  readily,  carefully  push  the 
coagulum  to  one  side  or  return  to  the  beakers.  Add  to  the  beakers  containing 
the  coagulum  50  c.  c.  of  0.1  percent  ammonium  sulphate  solution;  stir  for  one 
minute  and  decant  the  liquid  onto  the  filter.  Repeat  this  process  of  one-minute 
extractions  of  the  coagulum  in  0.1  percent  ammonium  sulphate  solution,  and 
filtration  as  above  directed,  until  the  filtrates  measure  about  450  c.  c.  Wash 
out  each  beaker  twice  with  8-10  c.  c.  of  hot  0.1  percent  ammonium  sulphate 
solution,  completing  the  transfer  of  the  coagulum  and  extract  to  the  sand. 


26 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


Wash  the  coagulum  twice  with  the  above  wash  solution  from  a  wash  bottle. 
At  all  times  allow  the  filter  to  drain  well  between  additions  of  extract  or  wash 
solution.  Combine  the  10  filtrates,  washing  out  the  container  of  each  of  the 
filtrates  twice  with  5-8  c.  c.  of  distilled  water.  Make  the  extract  up  to  5000  c.  c, 
and  mix. 

The  three  methods  compared,  for  the  precipitation  of  inorganic 
phosphorus  in  the  extracts  prepared  as  above,  and  for  the  final  esti- 
mation of  the  phosphorus,  were  as  specified  below : 

EMMETT  AND  GRINDLEY  NEUTRAL  AMMONIUM  MOLYBDATE 
METHOD  FOR  THE  DETERMINATION  OF  INORGANIC  PHOS- 
PHORUS IN  WATER  EXTRACTS  OF  FLESH 

Measure  out  the  number  and  volumes  of  extracts  indicated  in  the  schedule 
on  p.  24.  Evaporate,  with  frequent  stirring,  on  the  water  or  steam  bath  to 
approximately  20  to  25  c.  c.  While  hot,  filter  into  300  c.  c.  beakers,  using  doubled 
11  cm.  No.  589  (Blue  Ribbon  brand)  S.  and  S.  papers.  Wash  beakers,  precipi- 
tates, and  filters  thoroughly  with  hot  water.  The  volume  of  the  resulting  fil- 
trate' and  washings  should  be  about  125  c.  c.  Add  10  grams  of  ammonium 
nitrate  and  heat  upon  the  water  bath  to  60°  C.  Then  add  10  c.  c.  of  nitric  acid 
(sp.  gr.  1.20);  stir,  and  add  125  c.  c.  of  clear  ammonium  molybdate  solution. 
(Neutral  ammonium  molybdate  is  prepared  by  adding  ammonia  to  the  ordinary 
molybdate  solution,  using  litmus  paper  as  an  indicator.  This  work  should  be 
done  very  carefully,  and  both  red  and  blue  litmus  paper  used).  Reheat,  bring- 
ing temperature  to  60°  C.  Keep  at  this  temperature  for  15  minutes.  Stir 
vigorously  every  few  minutes  during  this  time.  Remove  from  the  bath  and 
allow  the  solutions  to  stand  2  hours  in  a  warm  place.  Decant  the  clear  super- 
natant liquid  through  doubled  11  cm.  No.  589  (Blue  Ribbon  brand)  S.  and  S. 
filters.  Transfer  the  remaining  liquid  and  precipitate  to  the  filters,  using  a 
10  percent  ammonium  nitrate  solution.  Wash  precipitates  and  beaker  four  or 
five  times  with  small  volumes  of  the  ammonium  nitrate  solution.  Dissolve  the 
yellow  precipitate  upon  the  filter,  and  that  in  the  precipitating  beaker,  with 
dilute  ammonium  hydroxid  (2.5  percent)  and  hot  water,  collecting  the  filtrate 
in  a  250  c.  c.  beaker.  Wash  thoroughly;  neutralize  the  solution  with  nitric  acid 
(1.20  sp.  gr.),  and  make  up  to  approximately  150  c.  c.  Add  5  grams  of  am- 
monium nitrate;  heat  upon  the  water  bath  to  60°  C.  and  then  carefully  add, 
while  stirring,  5  c.  c.  of  concentrated  nitric  acid  and  50  c.  c.  of  clear  acid 
molybdate  solution.  Digest  at  60°  C.  for  15  minutes,  stirring  occasionally. 
From  here  on  continue  the  determination  of  phosphorus  as  usual,  weighing  the 
phosphorus  as  magnesium  pyrophosphate. 

SIEGFRIED  AND  SINGEWALD  METHOD,  AS  USED  BY  EMMETT  AND 
GRINDLEY,  FOR  THE  DETERMINATION  OF  INORGANIC  PHOS- 
PHORUS IN  WATER  EXTRACTS  OF  ANIMAL  SUBSTANCES 

Measure  out  the  number  and  volumes  of  extracts  specified  in  the  schedule 
on  p.  24.  To  each  portion  add  50  c.  c.  of  a  10  percent  barium  chlorid  solution 
and  10  c.  c.  of  10  percent  ammonium  hydroxid.  Stir  the  solutions  every 
15  minutes  for  a  period  of  one  hour,  allow  to  stand  undisturbed  for  at 
least  12  hours,  and  then  filter  (decanting  at  first,  as  much  as  possible)  through 


INORGANIC  PHOSPHORUS  ESTIMATION  27 

double  quantitative  filters.  Wash  the  beakers,  precipitates  and  filters  repeat- 
edly, with  small  quantities  of  wash  water  containing  10  c.  c.  of  the  barium 
chlorid  solution  and  10  c.  c.  of  the  dilute  ammonium  hydroxid  per  liter.  Place 
the  upper  filters  containing  the  precipitates  in  the  beakers  in  which  the  precipi- 
tation occurred,  and  digest  at  room  temperature  with  35  c.  c.  of  dilute  nitric 
acid  (sp.  gr.  1.20)  with  frequent  stirring.  Filter  the  acid  solution  through 
the  second  filter,  which  was  not  removed  from  the  funnel,  and  wash  the  beakers 
and  filters  thoroughly  with  hot  water.  Neutralize  the  filtrates  with  ammonia, 
slightly  acidify  with  nitric  acid;  add  10  grams  of  ammonium  nitrate,  dilute 
to  about  125  c.  c.  and  heat  upon  the  water-bath  to  60^  C.  Add  100  c.  c.  of  acid 
ammonium  molybdate  and  continue  the  phosphorus  determination  from  here 
on  as  usual. 

MAGNESIA  MIXTURE  METHOD  OF  FORBES  AND  ASSOCIATES  FOR 

THE  DETERMINATION  OF  INORGANIC  PHOSPHORUS  IN 

EXTRACTS  OF  ANIMAL  TISSUES 

Measure  out  the  number  and  volumes  of  extracts  specified  in  the  schedule 
on  p.  24.  Add  10  c.  c.  magnesia  mixture,  stirring  freely;  allow  to  stand  15 
minutes,  and  add  25  c.  c.  of  ammonia,  sp.  gr.  0.90;  cover  and  allow  to  stand  over 
night. 

On  the  next  morning  filter,  and  wash  the  precipitate  with  2.5  percent 
ammonia  water.  Dissolve  the  precipitate  on  the  filter  paper  with  dilute  nitric 
acid  into  the  same  beaker  in  which  the  first  precipitation  was  made,  and  wash 
the  papers  thoroughly  with  hot  water.  Render  the  resulting  solutions  nearly 
neutral;  add  5  grams  of  ammonium  nitrate;  heat  to  65^  C;  add  50  c.  c.  of 
official  acid  molybdate  solution,  and  keep  at  60"  for  two  hours.  Then  continue 
in  the  usual  way  for  the  gravimetric  estimation  of  phosphorus  as  the  pjT:o- 
phosphate.     , 

The  numerical  results  of  this  comparative  study  of  methods  are 
reported  in  the  following  table,  these  data  being  discussed,  together 
with  those  from  the  work  of  1913,  on  pages  33-34. 


28 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


TABLE  XI:     COMPARISON  OF  METHODS  OF  ESTIMATION  OF 
INORGANIC  PHOSPHORUS  IN  ANIMAL  PRODUCTS 


Phos- 
phorus 
added 
(magne- 
sium 
pyro- 
phos- 
phate) 
Grams 

Magne- 
sium 
pyro- 
phos- 
phate 

obtained 

Grams 

Inor- 
ganic 
phos- 
phorus 

Percent 

Added  phosphorus 
recovered 

Sample 

Method,  and  sample 
number 

Volume 

of 

extract 

used 

c.  c. 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Percent 

Muscle 

Ai 

Neutral  Molybdate    A2 

Method  (Emmett      As 

and  Grindley)         A4 

Average 

500 
500 
250 
250 

0.0419 
0.0419 

0.0824 
0.0814 
0.0814 
0.0811 

0.5018 
0.4956 

0.4987 

0.0405 
0.0402 

0.0403 

96.2 

Muscle 

Bi 

Barium  Chlorid        B2 

Method  (Siegfried       Bs 

and  Singewald)        B4 

A  verage 

500 
500 
250 
250 

0.0419 
0.0419 

0.0836        0.5090 
0.0826        0.5029 
0.0850 
0.0848 

0.5059 

0.0435 
0.0433 

0.0434 

103.6 

Muscle 

Ci 

Magnesia  Mixture      C2 

Method   Forbes  and     C3 

Associates)            C4 

Average 

500 
500 
250 
250 

0.0419 
0.0419 

0.0792 
0.0794 
0.0798 
0.0806 

0.4822 
0.4834 

0.4828 

0.0402 
0.0410 

0.0406 

96.9 

Blood 

Ai 

Neutral  .Molybdate     A2 

Method  (Emmett      As 

and  Grindley)          A4 

A  verage 

500 
500 
250 
250 

0.0419 
0.0419 

0.0380 
0.0394 
0.0568 
0.0584 

0.0281 
0.0292 

0.0286 

0.0375 
0.0391 

0.0383 

91.4 

Blood 

Bi 

Barium  Chlorid         B2 

Method  (Siegfried        B;; 

and  Singewald)        B4 

Average 

500 
500 
250 
250 

0.0419 
0.0419 

0.0088 
0.0094 
0.0166 
0.0164 

0.0065 
0.0070 

0.0067 

0.0121 
0.0119 

0.0120 

28.6 

■       Ci 

Mag-nesia  Mi.xture      Cs 

•Ri^/t     !           Method  (Forbes  and     Cs 

^^°°^     1                    Associates)              C4 

Average 

500 
500 
250 
250 

0.0419 
0.0419 

0.0210 
0.0212 
0.0514 
0.0518 

0.0156 
0.0157 

0.0156 

0.0409 
0.0413 

0.0411 

98.1 

Ai 

Neutral  Molybdate     A 2 

Method  (Emmett       As 

Brain                    and  Grindley)           Ai 

A  verag'e 

500 
500 
250 
250 

0.0280 

0.0327 

0.0419        0.0540 

0.0419        0.0533 

0.0776 
0.0907 

0.0841 

0.0237 
0.0230 

0.0233 

55.6 

Brain 

Bi 

Barium  Chlorid        B2 

Method  (Siegfried       Bs 

and  Singewald)         B4 

A  verage 

500 
500 
250 
250 

0.0419 
0.0419 

0.0034 
0.0038 
0.0158 
0.0170 

0.0094 
0.0105 

0.0099 

0.0140 
0.0152 

0.0146 

34.6 

Brain 

Ci 

Magnesia  Mixture       C2 

Method  (Forbes  and     Cs 

Associates)             C4 

Average 

500 
500 
250 
250 

0.0419 
0.0419 

0.0216 
0.0210 
0.0498 
0.0507 

0.0599 
0.0582 

0.0590 

0.0392 
0.0401 

0.0396 

94.5 

Notes:    Weights  of  samples  used:  muscle  (vacuum  dried  sample)— 45.7822  grams:  blood  (fresh  pig's 
blood)— 376.2846  grams;  brain  (fresh  calf  brain)— 100.5238  grams;  extracts  made  up  to  5000  c  c 


INORGANIC  PHOSPHORUS  ESTIMATION 
THE  WORK  OF  1913 


29 


The  work  of  1913  consisted  of  a  further  comparison  of  the 
methods  of  Emmett  and  Grindley,  and  of  Forbes  and  associates,  with 
muscle,  blood  and  brain,  the  consideration  of  the  method  of  Siegfried 
and  Singewald  being  discontinued.  The  following  schedule  indicates 
the  estimations  made. 


1.  Muscle 

2.  Blood 

3.  Brain 


Extract  of  samples 
as  weighed 


B 
Extract  of  samples 
as  weighed  plus  25 
c.  c.  phosphate  sol- 
ution 


Ai  Neutral  molybdate  precipitation 

A  <<                            «                                  « 

A3 

Ai  Magnesia  mixture  precipitation 

A  «                                «                                        K 

A  «                            «                                  it 

Bi  Neutral  molybdate  precipitation 

Tin  "                                                 "                                                            " 

B.  "                 "                     " 

Bi  Magnesia  mixture  precipitation 

B„  "                 "                     " 


Phosphorus  was  also  determined  by  both  methods  of  precipita- 
tion in  the  phosphate  solution  used ;  and  blank  determinations  were 
made,  in  triplicate,  on  the  reagents. 


DIRECTIONS  FOR  THE  PREPARATION  OF  COLD  WATER 
EXTRACT  OF  MUSCLE 

A  Weigh  out  10-12  grams  of  fresh  muscle  and  divide  as  nearly  equally 
as  possible  between  two  small  beakers.  Moisten  the  samples  with  a  few  c.  c. 
of  distilled  water,  and  break  up  lumps  with  a  glass  rod.  Add  50  c.  c.  of  water 
to  each  beaker  and  stir  contents  for  15  minutes.  Allow  insoluble  residue  to 
settle  for  3-5  minutes;  then  decant  the  liquid  from  each  beaker  through  filters 
into  beakers;  allow  to  drain,  and  add  25  c.  c.  of  water.  Stir  for  7-8  minutes, 
and  after  allowing  to  settle,  decant  onto  the  same  filter.  Continue  this  treat- 
ment, using  each  time  25  c.  c.  of  water,  until  the  filtrates  measure  about  230  c.  c. 
each.  Allow  the  filters  to  drain  completely  between  extractions.  Whenever 
the  major  portion  of  the  residue  has  become  mechanically  transferred  to  the 
filter  return  it  to  the  beaker,  using  care  not  to  break  the  filter  paper.  After 
the  last  extraction  throw  the  entire  contents  of  each  beaker  onto  the  filter,  and, 
when  drained,  wash  twice  with  small  quantities  of  distilled  water.  Combine  the 
two  extracts,  and  use  for  the  precipitation  of  the  phosphates  under  A. 

B  Weigh  out  same  quantity  of  flesh  as  specified  above,  and  divide  as 
nearly  equally  as  possible  between  two  small  beakers;  work  up  with  a  few  c.  c. 
of  distilled  water;  add  25  c.  c.  of  aqueous  solution  of  disodium  phosphate, 
equivalent  to  about  40  mg.  magnesium  pyi'ophosphate,  dividing  as  nearly 
equally  as  possible  between  the  two  beakers,  and  proceed  as  directed  under  A. 
The  extract  thus  obtained  is  ready  for  precipitation  as  under  B. 


30  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

METHOD  FOR  THE  PREPARATION  OF  HOT  WATER-AMxMONIUM 
SULPHATE  EXTRACT  OF  BLOOD 

A  Weigh  out  30-35  grams  of  fresh  blood,  or  the  equivalent  of  oxalated 
blood,  into  a  400  c.  c.  beaker.  Add  a  few  c.  c.  of  distilled  water,  and  work  up 
the  blood  and  water  with  a  glass  rod.  Make  up  to  about  150  c.  c.  with  boiling 
distilled  water.  Place  over  a  flame,  and  gradually  bring  to  boiling,  with  con- 
stant stirring.  When  boiling  begins  add  20  c.  c.  of  20  percent  ammonium  sul- 
phate solution.  Boil,  with  constant  stirring,  for  about  ten  minutes.  Decant 
onto  a  filter  of  sand  on  linen,  receiving  the  filtrate  in  an  800  c.  c.  beaker.  When 
the  liquid  is  through,  lift  the  coagulum  from  the  sand,  and  transfer  it  to  a 
mortar.  Grind  to  a  smooth  paste  and  transfer  from  mortar  to  beaker  with 
boiling  distilled  water.  Make  up  to  about  50  c.  c.  with  the  same;  stir  for  8 
minutes  and  pour  contents  again  onto  the  sand  filter.  After  the  extract  is 
through,  return  the  coagulum  to  the  mortar,  and  grind  a  second  time,  trans- 
ferring to  the  beaker  as  before  with  boiling  distilled  water.  Repeat  this  process 
of  8-minute  extractions  of  the  coagulum  in  hot  water,  and  filtration  as  above 
directed,  without  further  grinding,  until  the  filtrate  measures  about  450  c.  c. 
Wash  out  each  beaker  twice  with  8-10  c.  c.  of  hot  water,  completing  the  trans- 
fer of  the  coagulum  and  extract  to  the  sand.  Wash  the  coagulum  on  the  sand 
twice  with  boiling  water  from  a  wash  bottle.  At  all  times  allow  the  filter  to 
drain  well  between  additions  of  extract  or  wash  w^ater.  This  extract  of  about 
500  c.  c.  is  ready  for  precipitation  as  under  A. 

B  Weigh  out  same  quantity  of  blood  as  specified  above.  Work  up  with 
a  few  c.  c.  of  distilled  water;  add  25  c.  c.  of  an  aqueous  solution  of  disodium 
phosphate  equivalent  to  about  40  mg.  magnesium  pyrophosphate,  and  proceed 
as  directed  under  A.  The  extract  thus  obtained  is  ready  for  precipitation 
under  B. 

METHOD  FOR  THE  PREPARATION  OF  HOT  WATER-AMMONIUM 
SULPHATE  EXTRACT  OF  BRAIN 

A  Weigh  out  about  ten  grams  of  brain  into  a  250  c.  c.  beaker.  Add  a  few 
c.  c.  of  distilled  water,  and  work  up  the  brain  and  water  with  a  glass  rod.  Make 
up  to  about  100  c.  c.  with  boiling  water;  place  over  a  flame,  and  gradually  bring 
to  boiling,  with  constant  stirring.  After  boiling  has  begun  add  20  c.  c.  of  20 
percent  ammonium  sulphate  solution;  boil  gently  for  about  ten  minutes;  allow 
to  settle  for  a  moment,  and  decant  liquid  slowly  onto  a  filter  of  sand  on  linen,* 
receiving  the  extract  in  an  800  c.  c.  beaker.  Add  to  the  beaker  containing  the 
coagulum  50  c.  c.  of  0.1  percent  ammonium  sulphate  solution.  Stir  for  one 
minute,  keeping  over  flame  and  at  the  boiling  point;  decant  the  liquid  onto  the 
filter.  Repeat  this  process  of  one-minute  extractions  of  the  coagulum  in  0.1 
percent  ammonium  sulphate  solution,  and  filtration  as  above  directed,  until  the 
filtrate  measures  about  450  c.  c.  Wash  out  the  beaker  twice  with  8-10  c.  c.  of 
hot  0.1  percent  ammonium  sulphate  solution,  completing  the  transfer  of  the 
coagulum  and  extract  to  the  sand.  Wash  the  coagulum  twice  with  the  above 
wash  solution  from  a  wash  bottle.  At  all  times  allow  the  filter  to  drain  well 
between  additions  of  extract  or  wash  solution.  This  extract  of  about  500  c.  c.  is 
ready  for  precipitation  under  A. 

*It  is  desirable  to  prevent  the  extract  or  coagulum  from  coming  in  contact  with  the  linen 
before  passing  through  the  sand.  To  this  end  pour  extract  slowly  onto  center  of  sand  or  into 
a  cup-shaped  depression. 


INORGANIC  PHOSPHORUS  ESTIMATION  31 

B  Weigh  out  same  quantity  of  brain  as  specified  above;  work  up  with  a 
few  c.  c.  of  distilled  water;  add  25  c.  c.  of  an  aqueous  solution  of  disodium 
phosphate  equivalent  to  about  40  mg.  of  magnesium  pyrophosphate,  and  pro- 
ceed as  directed  under  A.  The  extract  thus  obtained  is  ready  for  precipitation 
under  B. 

NEUTRAL  AMMONIUM  MOLYBDATE  METHOD  OF  EMMETT  AND 
GRINDLEY  FOR  THE  DETERMINATION  OF  INORGANIC  PHOS- 
PHORUS IN  WATER  EXTRACTS  OF  FLESH 

Treat  3  of  the  extracts  prepared  according  to  the  directions  on  the  pre- 
ceding pages  under  A,  and  3  of  those  prepared  according  to  B  as  follows: 
Evaporate,  with  frequent  stirring,  on  the  water  or  steam  bath,  to  approximately 
20-25  c.  c.  While  hot,  filter  into  300  c.  c.  beakers,  using  doubled  11  cm.  No.  589 
(Blue  Ribbon  brand)  S.  and  S.  papers.  Wash  beakers,  precipitates,  and  filters 
thoroughly  with  hot  water.  The  volume  of  the  resulting  filtrate  and  washings 
should  be  about  125  c.  c.  Add  10  grams  of  ammonium  nitrate  and  heat  upon 
the  water  bath  to  60°  C.  Then  add  10  c.  c.  of  nitric  acid  (sp.  gr.  1.20);  stir,  and 
add  125  c.  c.  of  clear  neutral  molybdic  solution.  (Neutral  ammonium  molybdate 
is  prepared  by  adding  ammonia  to  the  ordinary  molybdic  solution,  using  litmus 
paper  as  an  indicator.  This  work  should  be  done  very  carefully,  and  both  red 
and  blue  litmus  paper  used).  Reheat,  bringing  temperature  to  60°  C.  Keep 
at  this  temperature  for  15  minutes.  Stir  vigorously  every  few  minutes  during 
this  time.  Remove  from  the  bath  and  allow  the  solutions  to  stand  2  hours  in 
a  warm  place.  Decant  the  clear  supernatant  liquid  through  doubled  11  cm. 
No.  589  (Blue  Ribbon  brand)  S.  and  S.  filters.  Transfer  the  remaining  liquid 
and  precipitate  to  the  filters,  using  a  10  percent  ammonium  nitrate  solution. 
Wash  precipitates  and  beakers  four  or  five  times  with  small  volumes  of  the 
ammonium  nitrate  solution.  Dissolve  the  yellow  precipitate  upon  the  filter 
and  that  in  the  precipitating  beaker  with  dilute  ammonia  (2.5  percent)  and  hot 
water,  collecting  the  filtrate  in  a  250  c.  c.  beaker.  Wash  thoroughly.  Neutralize 
the  solution  with  nitric  acid  (1.20  sp.  gr.),  and  make  up  to  approximately  150 
c.  c.  Add  5  grams  of  ammonium  nitrate;  heat  upon  the  water  bath  to  60°  C. 
and  then  carefully  add,  while  stirring,  5  c.  c.  of  concentrated  nitric  acid  and 
50  c.  c.  of  clear  acid  molybdic  solution.  Digest  at  60°  C.  for  15  minutes,  stir- 
ring occasionally.  From  this  point  continue  the  determination  of  phosphorus 
as  usual,  weighing  the  phosphorus  as  magnesium  pyrophosphate. 

MAGNESIA  MIXTURE  METHOD  OF  FORBES  AND  ASSOCIATES  FOR 

THE  DETERMINATION  OF  INORGANIC  PHOSPHORUS 

IN  EXTRACTS  OF  ANIMAL  TISSUES 

Treat  three  of  the  extracts  prepared  according  to  the  directions  on  the  pre- 
ceding pages  under  A,  and  three  of  those  prepared  according  to  B  as  follows: 

Add  10  c.  c.  magnesia  mixture,  stirring  freely.  Allow  to  stand  15  minutes; 
add  25  c.  c.  ammonia,  sp.  gr.  0.90;  cover,  and  allow  to  stand  over  night.  On  the 
next  morning  filter,  and  wash  the  precipitate  with  2.5  percent  ammonia  water. 
Dissolve  the  precipitate  on  the  filter  paper  and  that  remaining  in  the  beaker 
in  which  the  precipitation  was  made  with  dilute  nitric  acid  (1:1)  and  hot  water, 
receiving  the  solution  in  400  c.  c.  beakers.  Neutralize  the  nitric  acid  with 
ammonia;  make  slightly  acid  with  nitric  acid;  add  5  grams  ammonium  nitrate, 
and  precipitate  in  the  usual  way  with  molybdate  solution.  Continue  in  the 
usual  way  for  the  gravimetric  estimation  of  phosphorus  as  the  p>Tophosphate. 


32 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


The  numerical  results  of  the  work  of  1913  are  reported  in  the 
following  group  of  tables,  these  data  being  discussed,  together  with 
those  from  the  work  of  1912,  on  pages  33  and  34. 


TABLE  XII:     COMPARISON  OF  METHODS  OF  ESTIMATION  OF 
INORGANIC  PHOSPHORUS  IN  ANIMAL  PRODUCTS 


Added 

Phos- 

phos- 

Magne- 

Inor- 

phorus 

phorus 

Added 

Blank 

Sample 

Treatment,  and 

Weight 
of 

sium 
pyro- 
phos- 
phate 

ganic 
phos- 

added 
(magne- 
sium 

recovered 
(magne- 
sium 

phos- 
phorus 

estima- 
tions 
on  rea- 

sample number 

sample 

phorus 

pyro- 
phos- 
phate) 

pyro- 
phos- 
phate) 

ered 

gents 

Grams 

Grams 

Percent 

Grams 

Grams 

Percent 

Grams 

Neutral  Moljbdate        Ai 

13.5485 

0.0276 

0.0568 

Neutral  Molybdate        A2 

11.5760 

0.0240 

0.0578 

Neutral  Molybdate        A3 

13.3025 

0.0259 

0,0543 

Average 

0.0563 

Same  plus  phosphate     Bi 

11.9395 

0.0648 

0.0407 

Same  plus  phosphate    Bl' 

11.5370 

0.0636 

0.0403 

Same  plus  phosphate     B3 

12.6880 

0.0654 

0.0398 

Muscle 

Average 

0.0417 

0.0403 

96.6 

Magnesia  Mixture          A  4 

14.5075 

0.0303 

0.0582 

Magnesia  Mixture          A  5 

10.0850 

0.0219 

0.0605 

Magnesia  Mixture          A  h 

11.4130 

0.0229 

0.0559 

Average 

0.0582 

Same  plus  phosphate    B4 

11.7065 

0.0617 

0.0373 

Same  plus  phosphate    B.i 

11.3530 

0.0606 

0.0369 

Same  plus  phosphate     Br, 

12.1045 

0.0621 

0.0368 

Average 

0.0417 

0.0370 

88.7 

Neutral  Molybdate       Ai 

29. 1950 

0.03241 

0.0309 

0.0020 

Neutral  Molybdate       A2 

35.0955 

0.03591- 

0.0285 

0.0016 

Neutral  Molybdate       A3 

28.4170 

0.02571 

0.0252 

0.0018 

Average 

0.0282 

0.0018 

Same  plus  phosphate    Bi 

36.5820 

0.07181 

0.0348 

Same  plus  phosphate    Bi' 

27.3368 

0.06621 

0.0385 

Same  pius  phosphate    B3 

32.8275 

0.06991 

0.0367 

Blood 

A  verage 

0.0417 

0.0367 

88.0 

Magnesia  Mixture          Ai 

30.9601 

0.01571 

0.0141 

0.0030 

Magnesia  Mixture          A5 

32.'<669 

0.01461 

0.0125 

0.0037 

Magnesia  Mixture          Ae 

29.2820 

0.01511 

0.0144 

0.0038 

Average 

0.0137 

0.0035 

Same  plus  phosphate    B4 

35.6872 

0.05451 

0.0370 

Same  plus  phosphate    B:, 

35.2757 

0.05651 

0.0392 

Same  plus  phosphate    Bi; 

36.1812 

0.05321 

0.0354 

Average 

ff.0417 

0.0372 

89.2 

Magnesia  Mixture          A4 

10.1650 

0.02461 

0.0575 

0.0010 

Magnesia  Mixture           A5 

10.3040 

0.02511 

0.0679 

0.0010 

Magnesia  Mixture          Ae 

10.9550 

0.02671 

0.0679 

0.0006 

Average 

0.0678 

0.0009 

Same  plus  phosphate    B4 

10.5095 

0.05121 

0.0257 

Same  plus  phosphate    Bs 

9. 2880 

0.04831 

0.0257 

Same  plus  phosphate    Be 

10.5465 

0.05141 

0.0258 

Brain 

Average 

0.0417 

0.0257 

96.6 

Magnesia  Mixture        2A4 

7.7011 

0.01761 

0.0662 

0.0008 

Magnesia  Mixture        2A,i 

9.2368 

0.02091 

0.0652 

0.0006 

Average 

0.0007 

Same  plus  phosphate   2B4 

10.7215 

0.05071 

0.0263 

Same  plus  phosphate   2E,^ 

9.2277 

O.O474I 

0.0264 

Same  plus  phosphate  2B(; 

10.5182 

0.05021 

0.0263 

Average 

0.0267 

0.0263 

98.9 

iBlanks  deducted. 


INORGANIC  PHOSPHORUS  ESTIMATION 


33 


TABLE  XIII:     TEST  OF  EFFECTS  OF  HEAT  AND  AMMONIUM 

SULPHATE  IN  THE  ESTIMATION  OF  INORGANIC 

PHOSPHORUS  IN   BLOOD 

Blood  of  Pig  Used;  Extracted  with  3.33  Percent  Ammonium  Sulphate 

Solution;  Each  Extract  About  500  c.  c.  in  Volume 


No.. 

Weight  of 
sample 

Treatment 

Magnesium 
pyrophosphate 

Inorganic 
phosphorus 

Grams 

Grams 

Percent 

1 

2 

Ave. 

33.0990 
34.4230 

Extract  evaporated;  boiled;  filtered 

0.0217 
0.0222 

0.0183 
0.0180 
0.0181 

3 

4 

Ave. 

33.3960 
31.3340 

Extract  evaporated;  filtered 

0.0216 
0.0218 

0.0180 
0.0194 
0.0187 

5 

6 

Ave. 

33.7026 
33.2960 

Extract  precipitated  directly 

0.0108 
0.0108 

0.0089 
0.0090 
0.0089 

CONCLUSIONS  FROM  WORK  OF  1912  AND  1913  ON  INORGANIC 
PHOSPHORUS  ESTIMATION  IN  ANIMAL  SUBSTANCES 

The  neutral  molybdate  method  of  Emmett  and  Grindley,  the 
barium  chlorid  method  of  Siegfried  and  Singewald,  (provided  a  suffi- 
cient excess  of  barium  chlorid  is  used)  and  the  magnesia  mixture 
method  of  Forbes  and  associates  all  gave  satisfactory  results,  which 
were  practically  identical,  on  vacuum-dried  muscle. 

The  barium  chlorid  method  was  found  inappHcable  in  the  pres- 
ence of  ammonium  sulphate,  and  hence  was  not  useful  on  extracts 
of  blood  and  brain  prepared  with  the  aid  of  this  reagent. 

The  neutral  molybdate  method  gave  results  on  blood  which  were 
apparently  too  high,  a  decomposition  of  organic  phosphorus  seeming 
to  result  from  the  heat  used  during  the  concentration  of  the  extract. 
Some  difficulty  was  experienced  in  the  recovery  of  inorganic  phos- 
phorus added  to  blood.  The  recovery  was  slightly  greater  with  the 
magnesia  mixture  than  with  the  neutral  molybdate  method. 

As  compared  with  the  magnesia  mixture  method  the  neutral 
molybdate  method  gave,  on  extract  of  brain,  prepared  with  the  aid 
of  ammonium  sulphate,  higher  results  for  inorganic  phosphorus, 
with  lower  recovery  of  added  phosphates,  (Table  XI,  p.  28).  The 
difficulties  of  filtration  are  greater  with  the  neutral  molybdate  than 
with  the  magnesia  m.ixture  method. 

Readily  filterable  extracts  of  brain  may  be  prepared  by  the  use 
of  3.33  percent  ammonium  sulphate  solution  in  place  of  0.1  percent 
ammonium  sulphate  in  each  place  where  the  latter  is  specified  in  the 


34  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

published  magnesia  mixture  method  (see  p.  25) ;  and  the  hinder- 
ing effect  of  the  added  amount  of  ammonium  sulphate  on  the  precipi- 
tation of  phosphorus  by  magnesia  mixture  may  be  overcome  by  the 
substitution  of  50  c.  c.  of  magnesia  mixture  for  the  10  c.  c.  as  speci- 
fied, and  allowing  the  precipitate  to  stand  3  days  before  filtering. 
With  these  modifications  the  magnesia  mixture  method  is  readily 
workable  on  brain ;  concordant  results  are  obtained,  and  added  phos- 
phate is  all  recovered.  The  work  on  brain  reported  in  Table  XII, 
p.  32,  was  done  by  this  modified  method. 

In  the  trial  reported  as  Samples  A4,  5  and  6,  and  B4,  5  and  6, 
Table  XII,  p.  32,  the  phosphate  was  all  recovered  but  0.0009  gm. 
magnesium  pyrophosphate.  In  the  trial  reported  as  Samples  2A4, 
5  and  6,  and  2B4,  5  and  6  the  loss  was  0.0004  gm.  magnesium  pyro- 
phosphate.    This  set  was  precipitated  in  a  refrigerated  room. 

The  neutral  molybdate  method  can  not  be  used  satisfactorily 
with  extracts  prepared  as  suggested  above,  with  the  aid  of  ammon- 
ium sulphate,  and  it  is  not  practicable  to  prepare  cold  water  extracts 
of  brain  as  in  the  neutral  molybdate  method,  which  has  been  used 
principally  with  flesh. 

The  test  of  the  influence  of  heat  on  inorganic  phosphorus  esti- 
mation in  blood,  as  set  forth  in  Table  XIII,  p.  33,  shows  that  the 
high  results  on  blood  obtained  by  the  neutral  molybdate  method 
must  be  due  to  the  cleavage  of  organic  phosphorus  by  the  heat  used 
in  the  evaporation  of  the  extract.  While  the  duration  of  heating 
used  in  this  method  is  much  greater  than  in  the  preparation  of  hot 
water-ammonium  sulphate  extracts  of  tissues  as  in  the  magnesia 
mixture  method,  this  test  raises  the  question  of  the  existence  and 
magnitude  of  such  cleavage.  This  point  has  received  further  con- 
sideration. 

The  recovery  of  added  phosphates  from  the  extract  of  muscle 
by  the  magnesia  mixture  method  is  usually  practically  complete  (see 
Table  XI,  p.  28).  In  the  last  analyses,  however,  (Table  XII,  p.  32), 
the  recovery  of  added  phosphate  was  appreciably  incomplete,  though 
the  determination  without  the  added  phosphate  was  higher  than 
by  the  neutral  molybdate  method,  where  the  recovery  of  added 
phosphate  was  practically  complete.  The  low  recovery  of  added 
phosphate  from  both  blood  and  muscle,  as  reported  in  this  table, 
suggests  that  the  conditions  were  not  perfect  for  the  precipitation 
of  this  amount  of  magnesium  ammonium  phosphate.  These  imper- 
fections were  ehminated  in  the  later  work.  With  brain  the  recovery 
of  added  phosphate  was  complete,  since  special  measures  (added 
amounts  of  magnesia  mixture,  and  increased  time  allowed  for  pre- 
cipitation) were  taken  to  insure  complete  precipitation. 


INORGANIC  PHOSPHORUS  ESTIMATION  85 

THE  WORK  OF  1914 

In  tlie  previous  work  on  inorganic  phosphorus  estimation  on 
animal  tissues  three  methods  have  been  compared,  namely,  the 
Neutral  Molybdate  Method  of  Emmett  and  Grindley,  the  modified 
Barium  Chlorid  Method  of  Siegfried  and  Singewald,  and  the  Mag- 
nesia Mixture  Method  of  Forbes  and  associates.  Satisfactory  com- 
parisons of  these  methods  have  been  made  on  muscle,  the  results, 
with  this  tissue,  being  practically  identical ;  and  certain  important 
limitations  to  the  applicability  of  the  two  methods  first  mentioned, 
to  tissues  other  than  muscle,  have  been  established.  It  was  now 
desired  to  test,  by  the  method  of  recovery  of  added  phosphates,  the 
accuracy  of  the  Magnesia  Mixture  Method,  in  its  latest  form,  with 
animal  tissues  of  diverse  character,  and  also  to  study  individually, 
a  number  of  details  of  this  method,  namely  (1)  the  influence  of  heat, 
as  specified,  (2)  the  method  of  filtration,  (3)  the  completeness  of 
extraction,  (4)  the  influence  of  ammonium  sulphate,  as  specified, 
(5)  the  effects  of  varying  amounts  of  ammonium  sulphate  and  (6) 
the  effects  of  diflferent  methods  of  use  of  ammonium  sulphate. 

In  the  test  of  the  accuracy  of  the  Magnesia  Mixture  Method, 
determinations  were  made,  in  triplicate,  on  blood,  brain,  flesh  and 
liver,  with  and  without  the  addition  of  known  amounts  of  inorganic 
phosphate.  The  detailed  directions  followed  in  this  test  are  on 
pages  35-37,  and  the  results  are  set  forth  in  Tables  XIV  and  XV 
on  pages  41  and  42.  The  results  of  the  further  analytical  proving 
of  the  details  of  this  method  were  all  made  on  blood.  The  data  are 
to  be  found-  in  Tables  XVI-XXI  on  pages  43  to  48. 

WATER-SOLUBLE  INORGANIC  PHOSPHORUS 
IN  ANIMAL  TISSUES 

From  samples  of  finely  divided  tissue  prepare  extracts  as  specified  under 
A,  B,  C  or  D,  and  determine  inorganic  phosphorus  as  specified  under  E. 

A.  PREPARATION  OF  COLD  WATER  EXTRACT  OF  FLESH 

Weigh  out  10-12  grams  of  fresh  muscle,  and  divide  as  nearly  equally  as 
possible  between  two  small  beakers.  Moisten  the  samples  with  a  few  c.  c.  of 
distilled  water,  and  break  up  lumps  with  a  glass  rod.  Add  50  c.  c.  of  water 
to  each  beaker  and  stir  contents  for  15  minutes.  Allow  insoluble  residue  to 
settle  for  3-5  minutes;  then  decant  the  liquid  from  each  beaker  through  filters 
into  beakers;  and  add  25  c.  c.  of  distilled  water  to  the  residue  in  the  beakers. 
Stir  for  7-8  minutes,  and  after  allowing  to  settle,  decant  onto  the  same  filter. 
Continue  this  treatment,  using  each  time  25  c.  c.  of  water,  until  the  filtrates 
measure  about  230  c.  c.  each.  Allow  the  filters  to  drain  completely  between 
extractions.  Whenever  the  major  portion  of  the  residue  has  become  mechanic- 
ally transferred  to  the  filter  return  it  to  the  beaker,  using  care  not  to  break 


36  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

the  filter  paper.  After  the  last  extraction  throw  the  entire  contents  of  each 
beaker  onto  the  filter,  and,  when  drained,  wash  twice  with  small  quantities 
of  distilled  water.  Combine  the  two  extracts,  and  determine  inorganic  phos- 
phorus as  under  E. 

B.  PREPARATION  OF  HOT  WATER-AMMONIUM 

SULPHATE  EXTRACT  OF  BLOOD 

Weigh  out  30-35  grams  of  fresh  blood,  (entire  portions  as  caught  from  the 
animal)  into  a  porcelain  mortar.  Grind  and  transfer  to  a  400  c.  c.  beaker  with 
hot  distilled  water.  Make  up  to  about  150  c.  c.  with  boiling  distilled  water. 
Place  over  a  flame,  and  gradually  bring  to  boiling,  with  constant  stirring. 
When  boiling  begins  add  20  c.  c.  of  20  percent  ammonium  sulphate  solution. 
Boil,  with  constant  stirring,  for  about  ten  minutes.  Decant  onto  an  18  cm.  filter 
paper,  receiving  the  filtrate  in  an  800  c.  c.  beaker.  When  the  liquid  is  through, 
lift  the  coagulum  from  the  paper,  being  very  careful  not  to  break  the  paper 
filter,  and  transfer  it,  along  with  that  remaining  in  the  beaker,  to  the  mortar. 
Grind  to  a  smooth  paste  and  transfer  from  mortar  to  beaker  with  boiling  3.33 
percent  ammonium  sulphate  solution.  Make  up  to  about  50  c.  c.  with  the  same, 
stir  for  8  minutes,  and  pour  contents  again  onto  the  filter  paper.  After  the 
extract  is  through,  return  the  coagulum  to  the  mortar  and  grind  a  second  time, 
transferring  to  the  beaker  as  before  with  boiling  3.33  percent  ammonium  sul- 
phate solution.  Repeat  this  process  of  8-minute  extractions  of  the  coagulum 
in  hot  3.33. percent  ammonium  sulphate  solution,  and  filtration  as  above  directed, 
without  further  grinding,  until  the  filtrate  measures  about  450  c.  c.  Wash  out 
each  beaker  twice  with  8-10  c.  c.  of  hot  3.33  percent  ammonium  sulphate  solu- 
tion, completing  the  transfer  of  the  coagulum  and  extract  to  the  filter  paper. 
Wash  the  coagulum  on  the  paper  twice  with  boiling  3.33  percent  ammonium 
sulphate  solution  from  a  wash  bottle.  At  all  times  allow  the  filter  to  drain 
well  between  additions  of  extract  or  wash  solution.  This  extract  of  about  500 
c.  c.  is  ready  for  precipitation  as  described  under  E. 

C.  PREPARATION  OF  HOT  WATER-AMMONIUM 

SULPHATE  EXTRACT  OF  LIVER 

Weigh  by  difi'erence  from  closed  weighing  bottles  15-20  gram  portions  of 
finely  ground  liver  into  400  c.  c.  beakers.  Add  a  few  c.  c.  of  cold  distilled  water, 
and  beat  up  with  a  stirring  rod  to  separate  the  particles  of  tissue.  Add  enough 
boiling  distilled  water  to  make  the  volume  about  150  c.  c;  place  over  a  flame 
and  bring  to  boiling.  Add  10  c.  c.  of  20  percent  ammonium  sulphate  solution, 
and  continue  to  boil  for  10  minutes. 

Remove  from  the  flame,  allow  to  settle,  for  a  moment  and  decant  the  boil- 
ing-hot liquid  onto  18  cm.  paper  filters.  Add  50  c.  c.  of  boiling  water  and  stir 
for  8  minutes,  without  further  heating  over  a  flame,  and  decant  onto  the  filter 
again.  Repeat  this  addition  of  50  c.  c.  of  hot  distilled  w^ater,  stirring,  and 
decanting  eight  times,  returning  the  coagulum  to  the  beaker  as  soon  as  any 
considerable  amount  collects  upon  the  filter.  With  the  eighth  portion  of  water 
throw  the  entire  contents  of  the  beaker  onto  the  filter  and  wash  twice  with  hot 
water  from  a  wash  bottle.  At  all  times  allow  the  filter  to  drain  well  between 
additions  of  extract  or  wash  water.  This  extract  of  about  600  c.  c.  is  now  ready 
for  precipitation  as  described  under  E. 


INORGANIC  PHOSPHORUS  ESTIMATION  37 

D.     PREPARATION  OF  HOT  WATER-AMMONIUM 
SULPHATE  EXTRACT  OF  BRAIN 

Weigh  out  about  ten  grams  of  brain  into  a  250  c.  c.  beaker.  Add  a  few 
cubic  centimeters  of  distilled  water,  and  work  up  the  brain  and  water  with  a 
glass  rod.  [Make  up  to  about  100  c.  c.  with  boiling  water;  place  over  a  flame, 
and  gradually  bring  to  boiling,  with  constant  stirring.  While  boiling  vigor- 
ously (not  before)  add  20  c.  c.  of  20  percent  ammonium  sulphate  solution;  boil 
gently  for  about  10  minutes;  allow  to  settle  for  a  moment,  and  decant  liquid 
slowly  onto  a  filter  of  acid-washed  glass  maker's  sand  on  linen,  receiving  the 
extract  in  an  800  c.  c.  beaker.  Add  to  the  beaker  containing  the  coagulum  50 
c.  c.  of  a  hot  3.33  pei-cent  ammonium  sulphate  solution.  Stir  for  one  minute, 
keeping  over  flame  and  at  the  boiling  point;  decant  the  liquid  onto  the  filter. 
Repeat  this  process  of  one-minute  extractions  of  the  coagulum  in  hot  3.33  per- 
cent ammonium  sulphate  solution,  and  filtration  as  above  directed,  until  the 
filtrate  measures  about  450  c.  c.  Wash  out  the  beaker  twice  with  8-10  c.  c.  of 
hot  3.33  percent  ammonium  sulphate  solution,  completing  the  transfer  of  the 
coagulum  and  extract  to  the  sand.  Wash  the  coagulum  twice  with  the  above 
wash  solution  from  a  wash  bottle.  At  all  times  allow  the  filter  to  drain  well 
between  additions  of  extract  or  wash  solution. 

This  extract  of  about  500  c.  c.  is  ready  for  precipitation  as  directed  under  E. 

PRECAUTIONS 

In  making  extracts  of  brain  it  is  desirable  that  the  analyst  give  careful 
attention  to  the  handling  of  the  sample.  The  coagulum  is  very  soft.  It  should 
be  stirred  only  enough  to  keep  it  in  motion.  If  roughly  handled  in  returning 
from  the  sand  filter  to  the  beaker  it  becomes  too  much  broken  up  and  holds 
onto  a  great  deal  of  liquid.  To  prevent  the  extract  or  the  coagulum  from 
coming  into  contact  with  the  linen  before  passing  through  the  sand  pour  the 
extract  slowly  into  a  slight  depression  in  the  center  of  the  sand,  or,  better  yet, 
onto  a  thin  film  of  absorbent  cotton  l^^  inches  in  diameter,  laid  over  a  de- 
pression in  the  sand.  ,The  coagulum  remains  on  the  cotton,  and  its  return  to- 
the  beaker  is  thereby  facilitated.  If  the  cotton  is  not  broken  up  by  needless 
stirring  it  can  be  taken  out  of  the  beaker  with  a  glass  rod  and  returned  to  the 
sand  each  time  a  partial  extract  is  to  be  filtered.  Care  is  necessary  to  prevent 
loss  through  bumping,  on  account  of  sand  in  the  beakers  during  the  last  extrac- 
tions.    Each  partial  extract  should  be  boiling  hot  at  the  time  filtration  begins. 

E.     MAGNESIA  MIXTURE  METHOD  FOR  THE  DETERMINATION 

OF  WATER-SOLUBLE  INORGANIC  PHOSPHORUS  IN 

EXTRACTS  OF  ANIMAL  TISSUES 

To  the  extracts  prepared  according  to  the  preceding  directions  add  50  c.  c. 
magnesia  mixture,  stirring  freely.  Allow  to  stand  15  minutes;  add  25  c.  c. 
ammonium  hydroxid,  sp.  gr.  0.90;  cover,  and  allow  to  stand  three  days.  Filter, 
and  wash  the  precipitate  with  2.5  percent  ammonia  water.  Dissolve  the  pre- 
cipitate on  the  filter  paper  and  that  remaining  in  the  beaker  in  which  the  pre- 
cipitation was  made  with  dilute  nitric  acid  (1:1)  and  hot  water,  receiving  the 
solution  in  400  c.  c.  beakers.  Neutralize  the  nitric  acid  with  ammonium 
hydroxid;  make  slightly  acid  with  nitric  acid.  Add  5  grams  ammonium  nitrate, 
and  precipitate  in  the  usual  way  with  molybdate  solution.  Continue  in  the  usual 
way  for  the  gravimetric  estimation  of  phosphorus  as  the  pyrophosphate. 


38  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

DISCUSSION  OF  RESULTS  OF  WORK  OF  1914 

The  data  in  Tables  XIV  and  XV,  pages  41  and  42,  show  that 
as  tested  by  the  recovery  of  added  phosphates,  the  Magnesia  Mix- 
ture Method,  in  the  form  stated  on  pages  35-37,  gives  results  appar- 
ently characterized  by  a  high  degree  of  accuracy.  The  recovery 
of  added  phosphates  was  96  percent  efficient  with  liver,  97  percent 
with  flesh,  99  percent  with  brain  and  100  percent  with  blood. 

In  consideration  of  the  close  agreement  of  triplicates,  the  high 
percentage  of  recovery  of  added  phosphates,  and  the  amounts  of 
coagulum  from  which  the  phosphates  were  recovered,  these  results 
are  considered  a  satisfactory  demonstration  of  the  reliability  of  the 
method. 

In  the  further  scrutiny  and  analysis  of  the  method,  however,  it 
was  deemed  advisable  to  test  individually  certain  of  its  details. 
Blood  was  selected  for  this  work,  since  the  ready  decomposition  of 
its  phosphocarnic  acid  was  considered  likely  to  reveal  possible  im- 
proprieties of  procedure.  The  results  of  these  studies  on  blood  are 
set  forth  in  Tables  XVI  to  XXI  on  pages  43-48. 

Table  XVI,  page  43,  gives  results  from  a  study  of  the  effects  of 
heat  and  ammonium  sulphate  in  this  estimation.  A  cold-water  ex- 
tract of  steer  blood  was  used.  This  extract  was  obtained  through 
the  use  of  a  centrifuge. 

In  sets  A  and  C  the  phosphates  were  precipitated  direct,  with 
magnesia  mixture,  with  and  without  ammonium  sulphate  added  (in 
the  cold)  before  precipitation.  The  results  were  practically  identi- 
cal, and  show  that,  in  the  cold,  ammonium  sulphate  does  not  affect 
inorganic  phosphate  determination  in  blood. 

Sets  B  and  D  were  boiled  containing  different  amounts  of  am- 
monium sulphate.  The  boiling  and  precipitation  of  inorganic  phos- 
phates in  1.25  percent  solution  of  ammonium  sulphate  (20  c.  c.  of 
20  percent  ammonium  sulphate,  as  specified)  gave  weights  of  mag- 
nesium pyrophosphate  half  of  a  milligram  greater  than  those 
obtained  from  boiling  and  precipitation  in  a  3.33  percent  solution 
of  ammonium  sulphate. 

These  results  were,  in  both  cases,  appreciably  lower  than  those 
obtained  from  A  and  C,  with  and  without  ammonium  sulphate,  but 
without  boiling.  These  results  show,  therefore,  that  ammonium 
sulphate,  in  the  cold,  is  without  influence  on  the  inorganic  phos- 
phorus estimation,  but  that  boihng  and  ammonium  sulphate  to- 
gether, as  used,  not  only  do  not  split  off  inorganic  from  organic 
phosphorus  compounds,  but,  as  shown  by  the  lower  results  obtained, 
cause  a  coagulation  and  precipitation  of  organic  phosphorus  in  the 


INORGANIC  PHOSPHORUS  ESTIMATION  39 

water  extract  which  when  not  so  precipitated  remains  in  solution 
until  precipitated  by  the  magnesia  mixture,  after  which  it  may  be 
hydrolyzed  by  nitric  acid  in  the  later  steps  of  the  phosphorus  esti- 
mation. 

Considering  the  possibility  that  the  lower  results  above  noted 
as  obtained  in  the  presence  of  ammonium  sulphate  might  be  due  to 
the  mechanical  inclusion  of  phosphates  in  the  coagulum,  another  set 
of  determinations  was  made,  as  reported  in  Table  XVII,  p.  44.  The 
grinding  of  the  coagulum  with  sand,  to  allow  of  more  complete  ex- 
traction and  washing,  gave  exactly  the  same  result  as  did  the  wash- 
ing of  the  coagulum  by  decantation,  in  the  usual  way.  Therefore, 
the  extraction,  as  usually  carried  out,  is  complete,  and  coagulation 
by  boiling  and  ammonium  sulphate  does  not  lock  up  inorganic  phos- 
phate by  mechanical  inclusion.  Further,  as  in  the  previous  set  of 
analyses,  lower  results  were  obtained  with  boiling  and  ammonium 
sulphate  than  with  direct  precipitation  in  the  cold,  though  the  re- 
covery of  added  phosphates  was  perfect  in  both  cases.  This  rein- 
forces our  previous  observation  as  to  the  precipitation  of  organic 
phosphorus  from  cold-water  extracts  of  blood,  along  with  the  inor- 
ganic phosphates.  Thus,  boiling  and  ammonium  sulphate  are 
needed  to  coagulate  a  certain  water-soluble  organic  phosphorus  frac- 
tion of  blood  in  the  estimation  of  inorganic  phosphorus  by  the 
Magnesia  Mixture  Method. 

The  results  in  Table  XVIII,  p.  45,  show  that  acid  alcohol  (0.2 
percent  nitric  acid)  will  dissolve  the  organic  phosphorus  which  is 
precipitated,  along  with  the  phosphates,  by  magnesia  mixture  alone, 
in  cold-water  extracts  of  blood ;  a  separation  of  the  organic  from  the 
inorganic  phosphorus  in  this  precipitate,  by  the  use  of  this  reagent, 
therefore,  is  not  possible. 

In  Table  XIX,  p.  46,  we  have  results  from  tests  made  to  deter- 
mine ( 1 )  whether  hot  water  or  ammonium  sulphate  should  be  used 
in  the  completion  of  the  extraction  of  the  coagulum  from  the  boiling 
with  ammonium  sulphate,  and  (2)  whether,  in  the  extraction  of 
blood,  the  partial  extracts  should  be  filtered  through  sand  on  linen 
or  through  filter  paper. 

Lower  results  (and,  therefore,  in  the  light  of  the  previous  evi- 
dence, more  nearly  correct  results)  were  obtained  when  a  3.33  per- 
cent solution  of  ammonium  sulphate,  rather  than  hot  water,  was 
used  in  the  completion  of  the  extraction  of  the  coagulum.  The  re- 
covery of  added  phosphates  was  also  higher  under  these  circum- 
stances. Filtration  of  the  blood  extracts  through  paper  was  found 
preferable  to  filtration  through  sand  on  linen. 


40  OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 

Table  XX,  p.  47,  reports  a  further  test  of  the  desirability  of 
using  ammonium  sulphate  in  the  completion  of  the  extraction  of  the 
coagulum  from  the  preliminary  boiling  with  ammonium  sulphate. 
As  in  the  previous  work  the  results  obtained  favored  the  use  of  the 
3.33  percent  solution,  since  this  procedure  led  to  lower  results  for 
inorganic  phosphorus  and  more  nearly  perfect  recovery  of  added 
phosphates. 

Table  XXI,  p.  48,  sets  forth  results  from  a  comparison  of  the 
use  of  different  amounts  of  ammonium  sulphate  in  the  coagulation 
and  extraction  of  blood.  No  advantage  could  be  demonstrated  as 
due  to  the  use  of  solutions  of  ammonium  sulphate  more  concentrated 
than  the  3.33  percent  solution  used  in  the  preceding  tests ;  that  is, 
the  use  of  3.33  percent  solutions  gave  lower,  and  apparently  more 
nearly  correct  results  than  were  obtained  with  a  1.25  percent  solu- 
tion, while  further  increase  of  the  concentration  of  the  ammonium 
sulphate  solution  did  not  lead  to  further  decrease  in  inorganic 
phosphate. 

CONCLUSIONS  FROM  WORK  OF  1914*  ON  INORGANIC  PHOS- 
PHORUS ESTIMATION  IN  ANIMAL  SUBSTANCES 

(1)  The  Magnesia  Mixture  Method  gives  satisfactorily  agree- 
ing results  on  blood,  brain,  liver,  and  flesh,  with  a  recovery  of  96-100 
percent  of  added  phosphates. 

(2)  Neither  ammonium  sulphate,  nor  boiling  and  ammonium 
sulphate  together,  as  used  in  the  Magnesia  Mixture  Method,  were 
found  to  cause  a  splitting  off  of  inorganic  from  organic  phosphorus 
in  blood. 

(3)  The  use  of  heat  and  ammonium  sulphate,  as  in  the  Mag- 
nesia Mixture  Method,  gives  lower  results  than  are  obtained  without 
heat  and  ammonium  sulphate,  though  the  recovery  of  added  phos- 
phates is  perfect ;  and  evidence  was  obtained  that  these  lower  results 
were  due  not  to  inclusion  of  phosphates  in  the  coagulum  obtained  by 
the  use  of  heat  and  ammonium  sulphate,  but  to  the  precipitation  of 
water-soluble  organic  phosphorus  compounds  which,  without  the 
use  of  heat  and  ammonium  sulphate,  yield  up  their  phosphorus  as 
inorganic  phosphate,  under  the  influence  of  the  nitric  acid  used  in 
the  subsequent  steps  of  the  inorganic  phosphorus  estimation. 

(4)  It  was  found  advisable  to  wash  the  coagulum  with  3.33  per- 
cent ammonium  sulphate  rather  than  with  hot  water.  A  more  con- 
centrated solution  was  shown  not  to  be  necessary. 

(5)  In  the  case  of  blood,  the  filtration  of  the  extract  through 
paper  was  found  preferable  to  the  filtration,  through  sand  on  linen, 
which  is  necessary  in  the  case  of  brain. 

*For  conclusions  from  the  work  of  1912  and  1913  see  pag-es  33  and  34. 


INORGANIC  PHOSPHORUS  ESTIMATION 


41 


(6)  The  methods  of  determination  of  inorganic  phosphorus  in 
blood,  brain,  flesh  and  liver  as  outlined  on  pages  35  to  37,  including 
the  details  of  extraction  as  well  as  of  actual  estimation  of  phos- 
phates, were  provisionally  adopted  in  1914  as  official  methods  of  the 
Association  of  Official  Agricultural  Chemists. 

For  conclusions  from  the  work  on  vegetable  substances  see 
page  21. 


TABLE  XIV:    TEST  OF  MAGNESIA  MIXTURE  METHOD  FOR  INORGANIC 

PHOSPHORUS  IN  ANIMAL  TISSUES  BY  RECOVERY 

OF  ADDED  PHOSPHATES 

A=Without  Phosphates      B— With  Added  Phosphates 


Added  phosphorus 

Phosphorus 

added 
(magnesium 

recovered 

Sample  and 

Weight 

Magnesium 

Inorganic 

determination 

of 

pj'rophos- 

phosphorus 

pyrophos- 

Magnesium 

No. 

sample 

phate 

phate) 

pyrophos- 
phate 

Percent 

Grams 

Grams* 

Percent 

Grams 

Grams 

Blood       Ai 

31.30 

0.0069 

0.00614 

A  2 

30.00 

0.0069 

0.00641 

As 

25.00 

0.0051 

0.00568 

Average 

0.00607 

Bi 

26.10 

0.0558 

0.0501 

B2 

28.20 

0.0563 

0.0501 

Bs 

30.50 

0.0572 

0.0505 

Average 

0.0497 

0.0502 

101.00 

Ai 

33.70 

0.0064 

0.00529 

A2 

33.60 

0.0060 

0.00505 

As 

31.20 

0.0060 

0.00544 

Averag'e 

0.00526 

Bi 

30.40 

0.0550 

0.0493 

B2 

32.20 

0.0557 

0.0496 

B3 

35.80 

0.0568 

0.0500 

Averag-e 

0.0496 

0.0496 

100.00 

*AU  blanks  deducted. 


42 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


TABLE  XV:     TEST  OF  MAGNESIA  MIXTURE  METHOD  FOR  INORGANIC 
PHOSPHORUS  IN  ANIMAL  TISSUES  BY  RECOVERY 
OF    ADDED    PHOSPHATES 
A=Without  Phosphates     B=With  Added  Phosphates 


Weight 
of  sample 

Grams 

Magnesium 
pyrophos- 
phate 

Grams* 

Inorganic 
phosphorus 

Percent 

Phosphorus 

added 
(magnesium 
pyrophos- 
phate) 

Grams 

Added  phosphorus 
recovered 

Sample  and 

determination 

No. 

Magnesium 
pyrophos- 
phate 

Graas 

Percent 

Brain          Ai 
Ao 
A3 

Average 
Bi 
B2 
B3 

Average 

8.5600 
7.7011 
9.2368 

10.7215 
9.2277 
10.5182 

Lost 
0.0176 
0.0209 

0.0507 
0.0474 
0.0502 

0.0636 
0.0630 
0.0633      - 

0.0266 

0.0263 
0.0264 
0.0263 
0.0263 

98.87 

Flesh          Ai 

A2 
A3 

Average 
Bi 
B2 
B3 

Average 

13.4653 
10.3444 
11.1769 

10.9638 
11.7942 
11.3154 

0.0272 
0.0207 
0.0223 

0.0694 
0.0725 
0.0708 

0.0562 
0.0557 
0.0555 
0.0558 

0.0496 

0.0474 
0.0488 
0.0481 
0.0481 

96.97 

Liver          Ai 

A2 
A3 

'  Average 

Bi 

B2 

B3 

Average 

16.2155 
13.8000 
14.4309 

14.9094 
14.9658 
16.2232 

0.0627 
0.0552 
0.0519 

0.1045 
0.1054 
0. 1094 

0.1077 
0.1144 
0. 1002 
0. 1064 

0.0496 

0.0475 
0.0482 
0  0474 
0.0477 

96.16 

*A11  blanks  deducted. 


INORGANIC  PHOSPHORUS  ESTIMATION 


43 


TABLE  XVI:    TEST  OF  EFFECTS  OF  BOILING  AND  VARYING  AMOUNTS 

OF  AiMMONIUM  SULPHATE  IN  THE  ESTIMATION  OF  INORGANIC 

PHOSPHORUS  IN  STEER  BLOOD  BY  THE  MAGNESIA 

MIXTURE  METHOD— COLD  WATER  EXTRACTS 


Sample 
No. 

Treatment 

Volume 
extract 

c.  c. 

Mag- 
nesium 
pyro- 
phosphate 
Gms. 

Phos- 
phorus 

Mgs. 

Ai 

A2 
A3 

Average 

Extract  precipitated  direct  with 
magnesia  mixture 

300 
300 
300 

0.0091 
0.0087 
0.0087 

2.535 
2.424 
2.424 

2.461 

Bi 

B2 
B3 

Average 

Extract  brought  to  boiling;  ammonium  sulphate 

added  to  make  1.25  percent  solution;  then 

boiled  for  ten  minutes 

300 
300 
300 

0.0079 
0.0078 
0.0081 

2.201 
2.173 
2.257 

2.210 

Ci 

C2 
C3 

Average 

Same   as   A,   with  ammonium  sulphate  to  make 

1.25  percent  solution  added  before 

precipitation 

300 
300 
300 

0.0085 
0.0085 
0.0086 

2.368 
2.368 
2.396 

2.374 

Di 
D2 

A  verage 

Same  as  B,  with  ammonium  sulphate  added  to 
make  3.33  percent  solution 

300 
300 
300 

0.0075 
0.0074 
0.0073 

2.090 
2.062 
2.034 

2.062 

All  of  the  above  extracts  and  filtrates  were  precipitated  by  adding  50  c.  c.  of  magnesia 
mixture  to  the  cool  solution,  and  then,  after  standing  a  short  period,  25  c.  c.  of  ammonia 
(sp.  gr.  .96). 


44 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


TABLE  XVII:     TEST  OF  COMPLETENESS  OF  EXTRACTION  AND 
EFFECTS  OF  BOILING  AND  AMMONIUM  SULPHATE  IN  THE 
ESTIMATION  OF  INORGANIC  PHOSPHORUS  IN  CALF 
BLOOD  BY  THE  MAGNESIA  MIXTURE  METHOD- 
COLD  WATER  EXTRACTS 


Volume 
of  ex- 
tract 

c.  c. 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Phos- 
phorus 
added 
(magne- 
sium 
pyro- 
phos- 
phate) 

Grams 

Added 
phosphorus 
recovered 

Sample 

No. 

Treatment 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Percent 

Al 

A  2 
A3 

Average 

Extract  precipitated  direct  with 
magnesia  mixture 

300 
300 
300 

0.0107 
0.0105 
0.0103 

0.0105 

A4 
As 
A6 

Average 

Same  as  A  l,  2,  3  +  25  c.  c.  of 
phosphate  solution 

300 
300 
300 

0.0619 
0.0618 
0.0620 

0.0619 

0.0517 

0.0514 

98.42 

Bi 
B2 
Bs' 

Average 

Extract  brought  to  boiling;  20  c.  c.  of  20^  am- 
monium sulphate  added,  and  boiled  for 
10  minutes,  filtered  and  washed 
by  decantation 

300 
300 
300 

0.0083 
0.0086 
0.0091 

0.0087 

B4 

Bs 
Be 

Average 

Same  as  Bi,  2,  3  but  coagulum  ground  with 

fine  sand  for  more  complete 

extraction  and  washing 

300 
300 
300 

0.0090 
0.0084 
0.0088 

0.0087 

Ci 

C2 
C3 

Average 

Same  as  Bi,  2,  3  + 25  c.  c. 
phosphate  solution 

300 
300 

300 

0.0603 
0.0601 
0.0604 

0.0603 

0.0517 

0.0516 

99.86 

C4 

Co 

C6 
Average 

Same  as  B4,  s,  6+25c.c. 
phosphate  solution 

300 
300 
300 

0.0601 
0.0609 
0.0602 

0.0604 

0.0517 

0.0517 

100.00 

All  of  the  above  extracts  and  filtrates  were  precipitated  by  adding  50  c.  c.  of  magnesia 
mixture  to  the  cool  solution,  and  then,  after  standing  a  short  period,  25  c.  c.  of  ammonia 
(.sp.  gr.  .96). 


INORGANIC  PHOSPHORUS  ESTIMATION 


45 


TABLE  XVIII:     TEST  OF  EFFECTS  OF  BOILING  AND  AMMONIUM  SUL- 

PHATE  IN  THE  ESTIMATION  OF  INORGANIC  PHOSPHORUS 

IN  STEER  BLOOD  BY  THE  MAGNESIA  MIXTURE 

METHOD— COLD   WATER   EXTRACTS 


Sample 
No. 

Treatment 

Volume 
of  extract 

c.  c. 

Mag- 
nesium 
pyro- 
phosphate 

Grams 

Phos- 
phorus 

Mg. 

Ai 
A2 
A3 

Average 

Extract  precipitated  direct  with 
magnesia  mixture 

200 
200 
200 

0.0075 
0.0070 
0.0070 

0.0072 

2.089 
1.950 
1.950 

1,996 

A4 
A5 
A6 

Average 

Extract  precipitated  as    Ai.  2.  sand   precipitate 
dissolved   in   acid   alcohol   (.2%  nitric   acid)    and 
phosphorus  determined  in  aliquots  of  this  solution 

200 
200 
200 

0.0076 
0.0073 
0.0065 

0.0071 

2.117 
2.034 
1.811 

1.987 

Bi 

B2 

B3 

Average 

Extract  boiled  for  20  minutes  with  20  c.  c.  of  20% 
ammonium    sulphate,  filtered    and    precipitated 
direct 

200 
200 
200 

0.0062 
0.0058 
0.0096* 

0.0060 

1.727 
1.616 

1.671 

*Precipitate  fused  during  ignition;   not  included   in   average. 

All  of  the  above  extracts  and  filtrates  were  precipitated  by  adding  50  c.  c.  of  magnesia 
mixture  to  the  cool  solution,  and  then,  after  standing  a  short  period,  25  c.  c.  of  ammonia, 
(sp.  gr.   .96). 


46 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


TABLE  XIX:     TEST  OF  METHODS  OF  USE  OF  AMMONIUM  SULPHATE 
AND  OF  METHODS  OF  FILTRATION  IN  THE  ESTIMATION  OF 
INORGANIC  PHOSPHORUS  IN  STEER  BLOOD  BY  THE 
MAGNESIA  MIXTURE  METHOD— HOT  WATER- 
AMMONIUM   SULPHATE  EXTRACTS 


Sample 
No. 

Treatment 

Weight 

of 
sample 

Grams 

Mag- 
nesium 
pyro- 
phos- 
phate 

Grams 

Phos- 
phorus 
added 
(mag- 
nesium 
pyrophos- 
phate) 

Grams 

Added 
phorus  re 

Mag- 
nesium 
pyrophos- 
phate 

Grams 

phos- 
covered 

Percent 

1 
2 
3 

Average 

Sample  extracted  in  usual  way  with  20  c.  c. 
of  20^  ammonium  sulphate;  filtered  through 
sand  on  linen 

34.2 
26.6 
30.7 

30.5 
1.0 

0.0079 
0.0061 
0.0064 

0.0068 
0.000223 

4 
5 
6 

Average 

Same  as  1,  2,  3  -j-  25  c.  c.  phosphate  solution 

30.7 
33.1 
38.2 

0.0583 
0.0582 
0.0590 

0.0537 
0.0537 
0.0537 

0.0515 
0.0508 
0.0505 

0.0509 

94.78 

7 

8 

•9 

Average 

Sample  extracted  as  usual;  then  subsequent 
extractions  made  with  3.33%  hot  ammonium 
sulphate;  filtered  through  sand  on  linen 

28.4 
34.2 
39.1 

33.9 
1.0 

0.0040 
0.0060 
0.0069 

0.0056 
0.000165 

10 
11 
12 

Average 

Same  as  7,  8,  9-|-25c.  c.  of  phosphate  solution 

37.6 
40.9 
32.4 

0.0590 
0.0590 
0.0576 

0.0537 
0.0537 
0.0537 

0.0528 
0.0522 
0.0522 

0.0524 

97.57 

13 
14 

Average 

Sample  extracted  usual  way,  +  25  c.  c.  phos- 
phate solution:   filtered    through    paper  in- 
stead of  sand  on  linen  (4,  5  and  6) 

32.6 
33.9 

0.0583 
0.0596 

0.0537 
0.0537 

0.0510 
0.0520 

0.0515 

95.90 

15 
16 

Average 

Sample  extracted  usual  waj'-r25  c.  c.  phos- 
phate solution;  subsequent  extraction  with 
3.33^       ammonium       sulphate;      filtration 
through  paper  instead  of  sand  on  linen  (10, 
11.  12) 

31.5 
37.4 

0.0600 
0.0603 

0.0537 
0.0537 

0.0548 
0.0541 

0.0544 

101.303 

The  above  filtrates  were  precipitated  by  adding  50  c.  c.  of  magnesia  mixture  to  the  cool 
solution,  and  then,  after  standing  a  short  period,  25  c.  c.  of  ammonia   (sp.  gr.  .96). 


INORGANIC  PHOSPHORUS  ESTIMATION 


47 


TABLE  XX:     TEST  OF  METHODS  OF  USE  OF  AMMONIUM  SULPHATE 

IN  THE  ESTIMATION  OF  INORGANIC  PHOSPHORUS  IN  STEER 

BLOOD  BY  THE  MAGNESIA  MIXTURE  METHOD— HOT 

WATER-AMMONIUM  SULPHATE  EXTRACTS 


Treatment 

Weight 

of 
sample 

Grams 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Phos- 
phorus 
added 
(magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Added 
phosphorus 
recovered 

Sample 
No. 

Magne- 
sium 
pyro- 
phos- 
phate 

Grams 

Percent 

Ai 

A2 

A3 
Average 

Sample  extracted  with  20  c.  c.  of  20*  am- 
monium sulphate  as  usual:  all  subse- 
quent extractions  made  with  hot  water 

32.0 
32.8 
34.6 

33.1 
1.0 

0.0078 
0.0080 
0.0083 

0.0080 
0.00024 

A4 

As 

Ae 
Average 

Same  as  Ai,  2.  3  +  25  c.c. 
phosphate  solution 

33.5 
34.1 
26.3 

31.3 

0.0573 
0.0569 
0.0556 

0.0566 

0.0496 

0.0491 

98.99 

Bi 

B2 
B3 

Average 

Same  as  Ai,  2.  3  only  all  subsequent  ex- 
tractions made  with  S.SS'/o  ammonium 
sulphate  instead  of  water 

33.7 
33.6 
31.2 

32.8 
1.0 

0.0064 
0.0060 
0.0060 

0.0061 
0.00019 

B4 
B5 

Be 
Average 

Same  as  Bi,  2.  3+25c.  c.  phosphate  solution 

30.4 
32.2 
35.8 

32.8 

0.0550 
0.0556 
0.0568 

0.0558 

0.0496 

0.0497 

100.20 

Blank  i 
3 

0.0005 
0.0005 
0.0004 

*A11   blanks   deducted. 

All  of  the  above  filtrates  were  precipitated  by  adding  50   c.   c.  magnesia  mixture  to  the 
cool  solution,  and  then,  after  standing  a  short  period,  25  c.  c.  of  ammonia  (sp.  gr.  .96). 


48 


OHIO  EXPERIMENT  STATION:  TECHNICAL  BUL.  8 


TABLE  XXI:  TEST  OF  VARYING  AMOUNTS  OF  AMMONIUM  SULPHATE 

IN  THE  ESTIMATION  OF  INORGANIC  PHOSPHORUS  IN  STEER 

BLOOD  BY  THE  MAGNESIA  MIXTURE  METHOD— HOT 

WATER-AMMONIUM    SULPHATE   EXTRACTS 


Sample 
No. 

Treatment 

Weight 

of 
sample 

Grams 

Mag- 
nesium 
pyro- 
phos- 
phate* 

Grams 

Phos- 
phorus 
added 
(mag- 
nesium 
pyro- 
phos- 
phate) 
Grams 

Added  phos- 
phorus recovered 

Mag- 
nesium 
pyro-     Percent 
phos- 
phate 
Grams 

Ai 

A2 
A3 

Average 

Sample  extracted  with  3.32'i  ammonium 
sulphate  throughout 

31.3 
30.0 
25.0 

28.76 
1.0 

0.0069 
0.0069 
0.0051 

0.0063 
0.00022 

A4 

As 
A6 

Average 

Same  as  Ai,  2,  3  +  25  c.  c.  phosphate 
solution 

26.1 
28.2 
30.5 

28.3 

0.0558 
0.0563 
0.0572 

0.0564 

0.0497        0.0502 

101.00 

Bi 

B2 

B3 

Average 

Sample  extracted  with  4i   ammonium 
sulphate  throughout 

27.8 
27.0 
30.8 

28.5 
1.0 

0.0071 
0.0069 
0.0074 

0.0071 
0.00025 

B4 

Bo 

Be 

Average 

Same  as  Bi,  2,  3  +  25  c.  c.  phosphate 
solution 

31.4 
31.2 
41.3 

34.6 

0.0584 
0.0579 
0.0576 

0.0580 

0.0497    1    0.0493        99.20 

Ci 
C2 

C3 

Average 

Sample  extracted  with  5%  ammonium 
sulphate  throughout 

28.1 
28.2 
30.5 

28.9 
1.0 

0.0065 
0.0070 
0.0067 

0.0067 
0.00023 

, 

C4 

Cs 
C6 

Average 

Same  as  Ci,  2.  3  +  25  c.  c.  phosphate  . 
solution 

30.5 
26.9 
45.5 

34.3 

0.0582 
0.0575 
0.0589 

0.0582 

0.0497        0.0503       101.21 

C7               500  c.  c.  of  5*  ammonium  sulphate -+- 
Cs                         25  c.  c.  phosphate  solution 

Average 

0.0498 
0.0496 

0.0497 

0.0497    j                    100.00 

All  of  the  above  filtrates  precipitated  by  adding  50 
solution,  and  then,  after  standing  for  a  short  period,  25  c. 
*A11  blanks  deducted. 


c.   magnesia  mixture  to   the   cool 
of  ammonia  (sp.  gr.  .96). 


COLUMBIA  UNIVERSITY  LIBRARIES 

This  book  is  due  on  the  date  indicated  below,  or  at  the 
expiration  of  a  definite  period  after  the  date  of  borrowing,  as 


provided  by  the  rules  of  the  Library  or  by  special  arrange-   i 
ment  with  the  Librarian  in  charge.                                                1 

DATE  BORROWED 

DATE  DUE 

DATE  BORROWED 

DATE  DUE 

c2e(n4t)Mioo 

QP535.P1 
Forbes 


F742 


